CN104611363A - Agrobacterium tumefaciens-mediated camelina sativa genetic transformation method - Google Patents
Agrobacterium tumefaciens-mediated camelina sativa genetic transformation method Download PDFInfo
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Abstract
The invention relates to an agrobacterium tumefaciens-mediated camelina sativa genetic transformation method which comprises the following steps: immersing camelina sativa inflorescences during an early flowering stage into agrobacterium tumefaciens liquid of a vector plasmid carrying a target gene, performing vacuum infiltration dip-dyeing and co-culture in a dark condition, recovering normal illumination culture of camelina sativa until T0-generation seeds are harvested, sowing the T0-generation seeds to obtain T1-generation plants, performing preliminary screening on the seeds or the plants by utilizing a marker gene in the plasmid, and finally identifying a camelina sativa transgenic plant carrying the target gene by utilizing a PCR (polymerase chain reaction) amplification technology and the like. Compared with a conventional dip-dyeing method, the method is relatively high in transformation rate, successfully realizes genetic transformation of the camelina sativa, and not only lays a foundation for application of a new transformed camelina sativa transgenic variety to the fields of functional foods, biofuel and the like but also provides a method and a technical reference for gene transformation of other types of plants.
Description
Technical field
The invention belongs to plant biotechnology field, especially a kind of Agrobacterium tumefaciens mediated butch flax genetic transforming method.
Background technology
Butch flax (Camelina sativa (L.) Crantz) belongs to Cruciferae, and having the good characteristics such as the Kang Han ﹑ Kang Han ﹑ ﹑ of Nai Ji Salt And Alkali Tolerance, disease and insect resistance, is a kind of oil crops of low input.Butch flax oil Linoleic acid content is 16% ~ 18%, and linolenic acid content is 36% ~ 40%, and natural tocopherol level is 42 ~ 48mg/100g, and polyunsaturated fatty acid total content is more than 60%, and unsaturated fatty acids total content is more than 90%.Therefore, butch flax oil, with its advantage such as natural health, chemically stable, is widely used in many fields such as high-grade health-care oil, foodstuff additive, medicine, makeup.
Simultaneously, along with fossil energy consumption manifesting the negative effect that unipolitics, energy economy & environment bring, the price volalility of fossil oil, the supply that political turbulence causes is unreliable, the factors such as greenhouse gas emission, finding the clean alternative renewable energy source of exploitation becomes urgent task.Because butch flax low cost, high yield, security are high by extensive concern, along with to its grease and grease methyl esters physico-chemical property and deepening continuously with its production biofuel feasibility analysis research, butch flax is identified as the huge emerging bio-fuel-oil crop of development potentiality.Fuel characteristic depends on the lipid acid composition preparing bio-fuel-oil raw material to a great extent, and biofuel requires higher resistance of oxidation, stability and combustioncharacteristics, and containing monounsaturated fatty acids oleic acid with height is more satisfactory composition.For solving scarcity of raw material problem, all the biodiesel plant kind being applicable to each department weather and ecological condition is actively being found in worldwide, therefore, modified by the hereditary property of method to butch flax of genetic transformation, for obtaining high yield, high gas oil ratio content, butch flax kind that fuel characteristic is good have important practical significance, and will promote China effectively in the research and development utilizing oilseed plant as the economical and effective energy.
In Genetic Transformation in Higher Plants, more traditional method that infects comprises: over-ground part is directly invaded in agrobacterium suspension and carries out infecting and directly dropping on bud with micro liquid sampler absorption agrobacterium suspension infecting.These two kinds of method transformation efficiencies are low, and easily pollute in dip-dye process.At present, the foundation about the genetic conversion system of butch flax rarely has report, and the butch flax genetic conversion system that therefore, construction procedures is simple, transformation efficiency is high, reproducible is still the problem needing solution in butch flax genetic improvement badly.
By patent retrieval, not yet find the patent publication us relevant to the present patent application patent.
Summary of the invention
The object of the invention is to the deficiency overcoming traditional butch flax genetic transfoumation, on the basis that tradition is contaminated, vacuum is adopted to contaminate conversion system, use General Instrument, the butch flax genetic conversion system that construction procedures is simple, transformation efficiency is high, reproducible, provide one with butch flax inflorescence for transformation receptor, by Agrobacterium tumefaciens mediated genetic transforming method, foreign gene is imported in butch flax recipient cell, through screening regeneration, obtains butch flax transfer-gen plant.
The technical scheme that the present invention realizes object is:
An Agrobacterium tumefaciens mediated butch flax genetic transforming method, described method is immersed in the agrobacterium tumefaciens bacterium liquid containing goal gene and screening-gene plasmid by the butch flax inflorescence come into bloom of pre-incubation, and concrete steps are as follows:
(1) the Construction and identification of engineering strain: adopt CaCl
2legal system for agrobacterium tumefaciens competent cell, average packing number pipe, be directly used in 24h conversion or liquid nitrogen flash freezer after-80 DEG C of preservations stand-by; Vector plasmid containing marker gene is imported in agrobacterium tumefaciens competent cell, often in pipe in agrobacterium tumefaciens competent cell: the volume ratio of YEB liquid nutrient medium is that the ratio of 1:8 adds YEB liquid nutrient medium, and mixing is placed on recovery 2h in 28 DEG C of environment; Centrifugal 45s under 4000 ~ 5000r/min room temperature, remove most of supernatant liquor, get the resuspended thalline of small part supernatant liquor, small part supernatant liquor is 1:1 with the volume ratio of every pipe agrobacterium tumefaciens competent cell, is applied to by resuspended thalline on the YEB solid resistant panel substratum containing 50mg/L Rifampin and 50mg/L kantlex; Be inverted for 28 DEG C and cultivate 24-48h, YEB solid resistant panel substratum obtains resistant clones;
The positive list bacterium colony of the Agrobacterium tumefaciens strain of random picking fresh culture from YEB solid resistant panel substratum, be inoculated in the YEB liquid nutrient medium containing the Rifampin of 50mg/L and the kantlex of 50mg/L, 28 DEG C, 180r/min shaking culture is spent the night; Alkali cracking method extracts plasmid, and after agarose gel electrophoresis detects, enzyme cuts qualification recombinant plasmid, gets the single bacterium colony of the successful agrobacterium tumefaciens of qualification conversion stand-by;
(2) the preparation of butch flax seedling to be transformed: by vermiculite, Nutrition Soil, perlite mixing, this mixture will ensure the nutrition of ventilation property and soil, evenly sow butch flax seed, overlay film 2-3d is with slow seedling, remain on 20/16 DEG C of (day/night) natural lighting plantation to cultivate, when the inflorescence of butch flax plant less than 20% be in completely open, more than 60% be in period in bud time for transforming;
(3) the preparation of vacuum infiltration medium: picking qualification transforms the single colony inoculation of successful agrobacterium tumefaciens in the YEB liquid nutrient medium containing 50mg/L Rifampin, and 28 DEG C of overnight incubation obtain bacterium liquid; Bacterium liquid being transferred to volume is in the above-mentioned YEB liquid nutrient medium of bacterium liquid 100 times itself, 28 DEG C, 180r/min sustained oscillation cultivation 24-48h, the centrifugal 10min of 4000-6000r/min, abandon supernatant, with above-mentioned YEB liquid nutrient medium: the volume ratio of aseptic osmotic medium is the aseptic osmotic medium suspension thalline of the ratio of 1:40-60, mix, namely prepare the permeating medium of transformed plant, stand-by;
(4) contaminate and Dual culture: butch flax comes into bloom, and plant is put into a moisture eliminator higher than 300mm, connects a vacuum pump, butch flax inflorescence is inverted and is immersed in the moisture eliminator filling above-mentioned permeating medium, vacuum pump evacuation keeps 85kPa to transform 5min; Then, entangle butch flax inflorescence with freshness protection package, put Dual culture 24-48h under dark condition, remove bag, recover to cultivate 20/16 DEG C of (day/night) natural lighting, until it is ripe to plant pod, results T
0for seed;
(5) the primary dcreening operation of transgenic seed or plant: utilize marker gene contained by vector plasmid to butch flax T
0for seed or sowing T
0the T cultivated for seed
1primary dcreening operation is carried out for plant;
(6) the qualification of transfer-gen plant: cultivate T
1for plant, with the plant leaf of the primary dcreening operation positive for material, extract plant DNA, for marker gene or goal gene design primer, adopt the qualification of PCR method whether can amplify goal gene fragment, if identify successfully, namely obtain butch flax transfer-gen plant.
And described step (1) middle vector plasmid is the pBG1100 with kalamycin resistance, and this plasmid contains the anti-careless fourth phosphine herbicide marker gene of Bar.
And described step concrete steps (5), are (6) as follows:
(5) the primary dcreening operation of transgenic seed or plant: containing the anti-careless fourth phosphine herbicide marker gene of Bar in vector plasmid pBinGlyBar1, as plant strain growth 3cm long to two leaf phases, stem, be that the ratio of 1:300 is mixed to get mixing solutions with volume ratio by careless fourth phosphine weedicide and water, use this mixing solutions to spray T
1for butch flax seedling; Within spraying herbicide one week, most of plant is curling by blade, withered until whole strain is dead, only a few plant still can survive, and getting can the plant of survive, namely completes the preliminary screening to transfer-gen plant;
(6) the qualification of transfer-gen plant: cultivate T
1for plant, with the plant leaf of the primary dcreening operation positive for material, extract plant template DNA, carry out PCR qualification for marker gene Bar gene design primer sequence 1/ sequence 2, the object fragment of 420bp can be amplified in transgenic line, namely obtain butch flax transfer-gen plant.
And described step (2) middle vermiculite, Nutrition Soil, perlite is the mixing of 2:2:1 ratio in mass ratio;
Or every L of described YEB solid resistant panel substratum consists of: 1.0g/L yeast extract paste+5.0g/L extractum carnis+5.0g/L peptone+0.5g/L MgSO
4+ 5.0g/L sucrose+15g/L agar+50mg/L Rifampin+50mg/L kantlex, pH 7.0-7.4.
And, being prepared as follows of described YEB solid resistant panel substratum: after being mixed by each component, pH is adjusted to 7.0-7.4,121 DEG C of sterilizing 20min, when substratum temperature is down to 60 DEG C, then adds Rifampin and kantlex, and it is stand-by that mixing is down flat plate.
And, described step (3) in aseptic osmotic medium consist of: the aqueous solution 5% (accounting for the mass percent of this substratum total mass) of 1/2MS substratum+sugar+efficient organic silicon surfactant 0.05% (accounting for the percent by volume of this substratum cumulative volume), pH 5.7.
And sugar is glucose, fructose, semi-lactosi, sucrose or lactose in the aqueous solution of described sugar.
And, being prepared as follows of described aseptic osmotic medium: the aqueous solution 5% (accounting for the mass percent of this substratum total mass) of 1/2MS substratum+sugar, pH is adjusted to 5.7, through 121 DEG C of sterilizing 20min, after temperature is down to normal temperature, add efficient organic silicon surfactant again, the nutrient solution after mixing is for the thalline that suspends.
And described step concrete steps are (5) as follows: if containing DSRed red fluorescent protein marker gene in vector plasmid, then green Led light transmission red sunglasses sheet is adopted to irradiate T
0for seed, what discernable by eye took on a red color fluorescence is transgenosis T
0for seed; If containing the anti-careless fourth phosphine herbicide marker gene of Bar in vector plasmid, then by T
1spray careless fourth phosphine weedicide for plant, can the preliminary judgement of normal growth be transfer-gen plant, carry out primary dcreening operation thus; Or, select other marker gene, the screening method that the different choice of the marker gene contained by vector plasmid is different.
And described agrobacterium tumefaciens is Agrobacterium tumefaciens strain EHA105.
Advantage of the present invention and beneficial effect are:
1, the inventive method utilizes molecular breeding technology, by RNA perturbation technique construction of expression vector plasmid, the method adopting vacuum infiltration to contaminate carries out genetic transformation to butch flax, cultivate the butch flax kind of high gas oil ratio content, make butch flax oil better meet the characteristic needs of biofuel, the production for biofuel provides reproducible high quality raw material.
2, the inventive method adopts vacuum to contaminate conversion system, and immersed by inflorescence in Agrobacterium penetrating fluid and vacuumize, under the pressure difference effect of front and back, bacterium liquid more easily enters inflorescence; Meanwhile, the efficient organic silicon surfactant Silwet L-77 in its osmotic medium has very strong absorption and penetrating power, contributes to bacterium liquid big area and is attached to for a long time on plant tissue, make Agrobacterium can enter vegetable cell smoothly; Sucrose, in conversion process, can maintain the stable of the outer pressure of pollen, provide energy simultaneously, can effectively improve Agrobacterium-mediated Transformation efficiency for Agrobacterium enters vegetable cell.
3, the inventive method required equipment is simple, operative technique is easily grasped, transformation efficiency is high, cost is low, the genetic transformation of butch flax can be realized, be not only transformation butch flax transgenosis new variety to lay a good foundation for the field such as functional foodstuff, biofuel, for foreign gene stably express in butch flax is laid a good foundation, also the reference on technique and method is provided for the genetic modification of other plants, be suitable for promotion and application, the genetic improvement of butch flax and molecular biology research tool are of great significance.
Accompanying drawing explanation
Fig. 1 is vector plasmid Hind III single endonuclease digestion electrophorogram, M:1kb DNA marker; G
2: former control plasmid digestion products; 1-3: the digestion products extracting plasmid;
Fig. 2 is the figure come into bloom on butch flax genetic transformation opportunity;
Fig. 3 is vacuum infiltration conversion system schematic diagram;
Fig. 4 is the PCR qualification figure of positive plant, M:DL 2000 DNA marker; 1 is positive control: the PCR primer of vector plasmid; 2 is negative control: the PCR primer of common unconverted plant; 3-8: the PCR primer transforming positive strain.
Embodiment
Below by specific embodiment, the invention will be further described, and following examples are descriptive, is not determinate, can not limit protection scope of the present invention with this.
Test method described in following embodiment, if no special instructions, is ordinary method; Described reagent and material, if no special instructions, all can obtain from commercial channels, or can prepare in conventional manner.
The butch flax inflorescence come into bloom immerses in the agrobacterium tumefaciens bacterium liquid of the vector plasmid carried containing goal gene by present method, contaminates after infiltration, Dual culture through vacuum, and butch flax plant recovers normal and cultivates, until results T
0for seed.Sowing T
0for seed, obtain T
1for plant, utilize the marker gene contained in plasmid to carry out preliminary screening to seed or plant, finally utilize the technical evaluation such as pcr amplification to carry the transfer-gen plant of goal gene.
Relevant operation steps in the present invention can be as follows:
CaCl
2legal system is for Agrobacterium EHA105 competent cell:
(1) Agrobacterium EHA105 is inoculated in sterilized 5mLYEB liquid nutrient medium, 28 DEG C of vibrations, 180r/min, incubated overnight;
(2) aseptically, moved into by 1mL overnight bacterial nutrient solution in 50mL YEB liquid nutrient medium, 28 DEG C of shaking culture are 0.4-0.6 to OD600, draw this bacterium liquid of 1mL and join in the sterile centrifugation tube of 2mL, place 5min on ice;
(3) under 4 DEG C of environment, with refrigerated centrifuge, the centrifugal 1min of 4500r/min, after supernatant discarded, is inverted centrifuge tube and blots nutrient solution as far as possible with aseptic filter paper;
(4) in centrifuge tube, add the 100m mol/L CaCl of 1mL precooling
2solution suspension thalline, blows and beats mixing gently with rifle head, ice bath 30min;
(5) again 4 DEG C, the centrifugal 1min of 4500r/min, supernatant discarded;
(6) the 100mmol/L CaCl of 100 μ L precoolings is added
2the resuspended precipitation of solution, competent cell is directly used in the sterile glycerol aqueous solution transforming or add 100 μ L 30%, and-80 DEG C store for future use.
Heat shock method transformation Agrobacterium:
L Agrobacterium EHA105 competent cell that () preserves from-80 DEG C of refrigerator taking-ups with glycerine, is positioned over 5min on ice immediately;
(2) vector plasmid getting 1-5ng extraction joins in the Agrobacterium EHA105 competent cell of 100 μ L, and mixing places 30min on ice;
(3) conversion fluid 42 DEG C of water-baths, thermal stimulus 50-90s, places 2min on ice;
(4) in conversion fluid, add the YEB liquid nutrient medium of 800 μ L, be put into 28 DEG C of constant-temperature table vibrations, activation culture 2h makes Agrobacterium recover;
(5) the centrifugal 1min of 10000r/min under room temperature, supernatant discarded, stay 100 μ L suspension thalline, abundant mixing, with aseptic spreader, bacterium liquid is applied in the resistant panel of the YEB solid medium containing 50mg/L kantlex (Kan) and 50mg/L Rifampin (Rif), be placed in 28 DEG C of constant-temperature bacterial culture box 1h, be inverted and cultivate 24-48h;
(6), after cultivating 24-48h, picking resistance list bacterium colony, upgrading grain carries out PCR or enzyme cuts qualification.
Embodiment 1
A kind of Agrobacterium tumefaciens mediated butch flax genetic transforming method, through the Construction and identification of the engineering strain carrying goal gene, the cultivation of butch flax seedling, the control of conversion opportunity, the preparation of vacuum infiltration device, engineering strain is made to contaminate butch flax, Dual culture, resistance screening, PCR detection, the final transfer-gen plant obtaining butch flax.
Described Agrobacterium tumefaciens mediated butch flax genetic transforming method, carries out as follows:
(1) Construction and identification of engineering strain: adopt CaCl
2legal system is for agrobacterium tumefaciens competent cell, and packing 100 μ L/ manages, and after being directly used in conversion or liquid nitrogen flash freezer in 24h ,-80 DEG C of preservations are stand-by.(vector plasmid is the pBG1100 with kalamycin resistance by vector plasmid to adopt heat shock method, this plasmid contains the anti-careless fourth phosphine herbicide marker gene of Bar) import in agrobacterium tumefaciens, add the Agrobacterium rhizogenes substratum YEB (Agrobacterium rhizogene Medium) of 800 μ L, mixing is placed on recovery 2h in 28 DEG C of environment.Centrifugal 45s under 4500r/min room temperature, removes most of supernatant 700 μ L, leaves and takes the resuspended thalline of 100 μ L residue supernatant and obtains resuspended thalline, be applied to by resuspended thalline in the YEB solid resistant panel containing 50mg/L Rif and 50mg/L Kan.Be inverted for 28 DEG C and cultivate 24-48h, resistant clones can be obtained at flat board.
The positive list bacterium colony of the agrobacterium strains EHA105 of random picking fresh culture YEB resistant panel, be inoculated into 25mL and contain in the YEB liquid nutrient medium of the Rifampin of 50mg/L and the kantlex of 50mg/L, 28 DEG C, 180r/min shaking culture is spent the night.After alkali cracking method extracts the detection of plasmid agarose gel electrophoresis, enzyme cuts qualification recombinant plasmid, the results are shown in Figure 1.
Described YEB solid resistant panel substratum consists of: 1.0g/L yeast extract paste+5.0g/L extractum carnis+5.0g/L peptone+0.5g/L MgSO
4+ 5.0g/L sucrose+15g/L agar+50mg/L Rif+50mg/L Kan, pH 7.0-7.4.During preparation, pH is adjusted to 7.0-7.4,121 DEG C of autoclaving 20min, when substratum temperature is down to about 60 DEG C, then adds Rif and Kan, and it is stand-by that mixing is down flat plate.
Need resistance screening owing to transforming bacterial strain, that the vector plasmid described in the present embodiment is selected is the pBinGlyBar1 with Kan resistance.Other any DNA or plasmids that can transform butch flax, also can adopt method of the present invention to transform.
(2) cultivation of butch flax seedling: vermiculite, Nutrition Soil, perlite are mixed in the ratio of 2:2:1, evenly sow butch flax seed, overlay film 2d is with slow seedling, remain on 20/16 DEG C of (day/night) natural lighting plantation to cultivate, when the inflorescence of butch flax plant less than 20% be in completely open, more than 60% be in period in bud time for transforming.
(3) preparation of vacuum infiltration medium: picking qualification transforms the single colony inoculation of successful Agrobacterium in the 5mLYEB liquid nutrient medium containing 50mg/L Rifampin, 28 DEG C of overnight incubation.Be transferred to by bacterium liquid in the identical YEB nutrient solution of 500mL, 28 DEG C, 24-48h is cultivated in 180r/min sustained oscillation.The centrifugal 10min of 6000r/min, abandons supernatant, and with 300mL aseptic osmotic medium suspension thalline, mix, the permeating medium preparing transformed plant is stand-by.
Described osmotic medium consists of: 1/2MS (MS is the name abbreviation of Murashige and Skoog two people of this substratum of design)+aqueous sucrose solution 5% (m/v)+efficient organic silicon surfactant (Silwet-L77) 0.05% (volume fraction), pH 5.7.During preparation, 1/2MS+ aqueous sucrose solution 5% (m/v), pH is adjusted to 5.7, and through 121 DEG C of autoclaving 20min, after temperature is down to normal temperature, then add silwet L-77 tensio-active agent, the nutrient solution after mixing is for the thalline that suspends.
(4) dip-dye and Dual culture: butch flax comes into bloom, plant is put into the high moisture eliminator of a 310mm, connect a vacuum pump, butch flax inflorescence is inverted and is immersed in the beaker filling the above-mentioned permeating medium of 300mL, vacuum pump evacuation keeps 85kPa to transform 5min.Entangle butch flax inflorescence with freshness protection package, put Dual culture 24h under dark condition, remove bag, recover normal illumination, until results T
0for seed.
The conversion opportunity of described butch flax seedling is for coming into bloom, i.e. the less opening of butch flax inflorescence, major part is for being used for when a bud just ready to burst transforming.
(5) primary dcreening operation of transgenic seed or plant: utilize marker gene contained by vector plasmid to butch flax T
0for seed or sowing T
0the T cultivated for seed
1primary dcreening operation is carried out for plant.
Described prescreening method, the different marker gene contained by plasmid is specifically selected.Containing the anti-careless fourth phosphine herbicide marker gene of Bar in the plasmid pBinGlyBar1 selected in the present embodiment, therefore, careless fourth phosphine weedicide can be used T
1primary dcreening operation is carried out for butch flax seedling.Equally, if containing red fluorescent protein marker gene in the plasmid selected, then green Led light transmission red sunglasses sheet can be adopted to irradiate T
0for seed, discernable by eye goes out transgenosis T
0primary dcreening operation is carried out for seed.Select the vector plasmid carrying good riddled basins particularly important, experiment progress can be accelerated, shorten the screening cycle, effectively increase work efficiency.
(6) qualification of transfer-gen plant: cultivate T
1for plant, with the plant leaf of the primary dcreening operation positive for material, extract plant DNA, at expression vector G
2on to choose Bar gene fragment be template, utilize VectorNTI to design primer, design of primers result is as shown in table 1, adopts PCR method to identify primary dcreening operation positive plant.
Table 1 Bar design of primers
Take DNA as template, with b-F and b-R sequence for primer, carry out pcr amplification, reaction system is 20 μ L, and concrete composition is in table 2.Response procedures: 95 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 2min, 35 circulations; 72 DEG C extend 10min.After reaction terminates, sample 3 μ L and adopt 1% agarose gel electrophoresis, detect whether amplify object fragment, the results are shown in Figure 4.
Table 2 Bar gene PCR amplification system
Embodiment 2
A kind of Agrobacterium tumefaciens mediated butch flax genetic transforming method, through the Construction and identification of the engineering strain carrying containing goal gene, the cultivation of butch flax seedling, the control of conversion opportunity, the preparation of vacuum infiltration device, then engineering strain is made to contaminate butch flax, Dual culture, resistance screening, PCR detection, the transfer-gen plant of final acquisition butch flax, concrete steps are as follows:
One, the Construction and identification of engineering strain
(1) CaCl is adopted
2legal system is for agrobacterium tumefaciens competent cell, and packing 100 μ L/ manages, and after being directly used in conversion or liquid nitrogen flash freezer in 24h ,-80 DEG C of preservations are stand-by;
(2) adopt heat shock method to be imported in agrobacterium tumefaciens competent cell by vector plasmid pBinGlyBar1, add the YEB substratum of 800 μ L, mixing is placed on recovery 2h in 28 DEG C of environment; Centrifugal 45s under 4500r/min room temperature, remove most of supernatant liquor 700 μ L, leave and take 100 μ L and remain the resuspended thalline of supernatant liquor, resuspended thalline is applied in the YEB solid resistant panel containing 50mg/L Rifampin and 50mg/L kantlex, be inverted for 28 DEG C and cultivate 24-48h, resistant clones can be obtained at flat board;
YEB solid resistant panel substratum consists of: 1.0g/L yeast extract paste+5.0g/L extractum carnis+5.0g/L peptone+0.5g/LMgSO
4+ 5.0g/L sucrose+15g/L agar+50mg/L Rifampin+50mg/L kantlex, pH 7.0-7.4, during preparation, pH is adjusted to 7.0-7.4,121 DEG C of sterilizing 20min, when substratum temperature is down to about 60 DEG C, then adds Rif and Kan, and it is stand-by that mixing is down flat plate;
(3) positive list bacterium colony of the agrobacterium strains EHA105 of random picking fresh culture YEB resistant panel, being inoculated into 25mL contains in the YEB liquid nutrient medium of the Rifampin of 50mg/L and the kantlex of 50mg/L, 28 DEG C, 180r/min shaking culture is spent the night;
(4) alkali cracking method extracts plasmid, owing to adopting the restriction enzyme site of Hind III restriction enzymes in the vector plasmid of structure containing 4, therefore, this cyclic plasmid is through the gel electrophoresis of Hind III single endonuclease digestion, calculate according to known plasmid map, the single band (as shown in Figure 1) that four sizes are known can be obtained.Digestion products electrophoresis result shows that engineering strain successfully constructs.
Need resistance screening owing to transforming bacterial strain, that vector plasmid used in the present invention is selected is the pBinGlyBar1 with Kan resistance.Other any DNA or plasmids that can transform butch flax, also can adopt method of the present invention to transform.
Two, the butch flax genetic transformation that engineering bacteria is Agrobacterium tumefaciens mediated
(1) preparation of vacuum infiltration medium: picking identifies that successful engineering strain list colony inoculation is in the 5mLYEB liquid nutrient medium containing 50mg/L Rif and 50mg/L Kan, 28 DEG C of overnight incubation.Be transferred to by bacterium liquid in the identical YEB nutrient solution of 500mL, 28 DEG C, 24-48h is cultivated in 180r/min sustained oscillation.The centrifugal 10min of 6000r/min, abandons supernatant, with 300mL aseptic osmotic medium suspension thalline, mixes, namely prepares the permeating medium of transformed plant, stand-by.
Osmotic medium consists of: 1/2MS+ aqueous sucrose solution 5% (m/v)+silwet L-77 tensio-active agent 0.05% (v/v), pH 5.7.During preparation, 1/2MS+ aqueous sucrose solution 5% (m/v), pH is adjusted to 5.7, and through 121 DEG C of sterilizing 20min, after temperature is down to normal temperature, then add silwet L-77 tensio-active agent, the nutrient solution after mixing is for the thalline that suspends.
(2) dip-dye and Dual culture: by vermiculite, Nutrition Soil, perlite mixes in the ratio of 2:2:1, evenly sow butch flax seed, overlay film 2d is with slow seedling, remain on 20/16 DEG C of (day/night) natural lighting plantation to cultivate, when the inflorescence of butch flax plant less than 20% is in opening completely, more than 60% when being in period in bud for transforming, conversion opportunity is that butch flax comes into bloom (as shown in Figure 2), plant is put into the high moisture eliminator of a 310mm, connect a vacuum pump, butch flax inflorescence is inverted to be immersed in the beaker filling the above-mentioned permeating medium of 300mL, vacuum pump evacuation keeps 85kPa to transform 5min (as shown in Figure 3).Then, entangle butch flax inflorescence with freshness protection package, put Dual culture 24h under dark condition, remove bag, recover normal illumination and cultivate, until results T
0for seed.
(3) primary dcreening operation of transgenic seed or plant: containing the anti-careless fourth phosphine herbicide marker gene of Bar in vector plasmid pBinGlyBar1, therefore, plant strain growth to two leaf phases, during long about the 3cm of stem, careless fourth phosphine medicament is mixed with the ratio of 1:300 with water, uses this mixing solutions to spray T
1for butch flax seedling.Within spraying herbicide one week, most of plant is curling by blade, withered until whole strain is dead, only a few plant still can survive, substantially can complete the preliminary screening to transfer-gen plant.
(4) qualification of transfer-gen plant: cultivate T
1for plant, with the plant leaf of the primary dcreening operation positive for material, extract plant template DNA.PCR qualification is carried out for marker gene Bar gene design primer 5 '-GCTGAAGTCCAGCTGCCAGAAAC-3 '/5 '-GAGACAAGCACGGTCAACTTCC-3 ', the object fragment (as shown in Figure 4) of 420bp can be amplified as a result in 6 transgenic lines, result shows, expression vector successfully proceeds in butch flax, obtains 6 transgenic lines.
Embodiment 3
An Agrobacterium tumefaciens mediated butch flax genetic transforming method, through carrying the Construction and identification of the engineering strain containing goal gene, concrete steps are as follows:
(1) CaCl is adopted
2legal system is for agrobacterium tumefaciens competent cell, and packing 100 μ L/ manages, and after being directly used in conversion or liquid nitrogen flash freezer in 24h ,-80 DEG C of preservations are stand-by;
(2) adopt heat shock method to make vector plasmid pBinGlyBar1 transform Agrobacterium tumefaciens, add the YEB substratum of 800 μ L, mixing is placed on recovery 2h in 28 DEG C of environment; Centrifugal 45s under 4500r/min room temperature, remove most of supernatant 700 μ L, leave and take 100 μ L and remain supernatant resuspended thalline, resuspended thalline is applied in the YEB solid resistant panel containing 50mg/L Rifampin and 50mg/L kantlex, be inverted for 28 DEG C and cultivate 24-48h, obtain resistant clones at flat board;
Heat shock method transformation Agrobacterium: the Agrobacterium EHA105 competent cell preserved with glycerine from-80 DEG C of refrigerator taking-ups, is positioned over 5min on ice immediately; Get the vector plasmid G that 5 μ L extract
2join in the Agrobacterium EHA105 competent cell of 100 μ L, mixing, places 30min on ice; Conversion fluid is proceeded to 42 DEG C of water bath with thermostatic control 90s, then place 2min on ice, add the YEB liquid nutrient medium of 800 μ L in conversion fluid, mixing is placed on shaking table 28 DEG C of constant-temperature shaking culture, activation culture 2h;
Described YEB solid resistant panel substratum consists of: 1.0g/L yeast extract paste+5.0g/L extractum carnis+5.0g/L peptone+0.5g/L MgSO
4+ 5.0g/L sucrose+15g/L agar+50mg/L Rifampin+50mg/L kantlex, pH 7.0-7.4, during preparation, pH is adjusted to 7.0-7.4,121 DEG C of sterilizing 20min, when substratum temperature is down to about 60 DEG C, then adds Rif and Kan, and it is stand-by that mixing is down flat plate;
(3) positive list bacterium colony of the agrobacterium strains EHA105 of random picking fresh culture YEB resistant panel, being inoculated into 25mL contains in the YEB liquid nutrient medium of the Rifampin of 50mg/L and the kantlex of 50mg/L, 28 DEG C, 180r/min shaking culture is spent the night;
(4) alkali cracking method extracts plasmid, owing to adopting the restriction enzyme site of Hind III restriction enzymes in the vector plasmid of structure containing 4, therefore, this cyclic plasmid can adopt Hind III restriction enzyme single endonuclease digestion, and enzyme cuts system as table 3,37 DEG C of water-baths, reaction 3h, whether digestion products adopts 1% agarose gel electrophoresis to detect, analyze consistent with expection band, thus whether qualification engineering strain successfully constructs.
Table 3 endonuclease reaction system
Restriction enzyme kind contained by different plasmid and the difference of number, determine that different Restriction enzyme combinations is identified engineering strain.
Need to carry out resistance screening owing to transforming bacterial strain, that the vector plasmid described in the present embodiment is selected is the pBinGlyBar1 with Kan resistance.Other any DNA or plasmids that can transform butch flax, also can adopt method of the present invention to transform.
(5) cultivation of butch flax seedling: vermiculite, Nutrition Soil, perlite are mixed in the ratio of 2:2:1, evenly sow butch flax seed, overlay film 2d is with slow seedling, remain on 20/16 DEG C of (day/night) natural lighting plantation to cultivate, when the inflorescence of butch flax plant less than 20% be in completely open, more than 60% be in period in bud time for transforming.
(6) preparation of vacuum infiltration medium: picking qualification transforms the single colony inoculation of successful Agrobacterium in the 5mLYEB liquid nutrient medium containing 50mg/L Rifampin, 28 DEG C of overnight incubation.Be transferred to by bacterium liquid in the identical YEB nutrient solution of 500mL, 28 DEG C, 24-48h is cultivated in 180r/min sustained oscillation.The centrifugal 10min of 6000r/min, abandons supernatant, and with 300mL aseptic osmotic medium suspension thalline, mix, the permeating medium preparing transformed plant is stand-by.
Described osmotic medium consists of: 1/2MS (MS is the name abbreviation of Murashige and Skoog two people of this substratum of design)+aqueous sucrose solution 5% (m/v)+efficient organic silicon surfactant (Silwet-L77) 0.05% (volume fraction), pH 5.7.During preparation, 1/2MS+ aqueous sucrose solution 5% (m/v), pH is adjusted to 5.7, and through 121 DEG C of autoclaving 20min, after temperature is down to normal temperature, then add silwet L-77 tensio-active agent, the nutrient solution after mixing is for the thalline that suspends.
(7) dip-dye and Dual culture: as shown in Figure 3, butch flax comes into bloom, and plant is put into the high moisture eliminator of a 310mm, connects a vacuum pump, butch flax inflorescence is inverted to be immersed in the beaker filling the above-mentioned permeating medium of 300mL, and vacuum pump evacuation keeps 85kPa to transform 5min.Then, entangle butch flax inflorescence with freshness protection package, put Dual culture 24h under dark condition, remove bag, recover normal illumination, until results T
0for seed.
The conversion opportunity of described butch flax seedling is for coming into bloom, i.e. the less opening of butch flax inflorescence, major part, for being used for when a bud just ready to burst transforming, is shown in Fig. 2.
(8) primary dcreening operation of transgenic seed or plant: utilize marker gene contained by vector plasmid to butch flax T
0for seed or sowing T
0the T cultivated for seed
1primary dcreening operation is carried out for plant.
Described prescreening method, contained by plasmid, different marker gene is specifically selected.Containing the anti-careless fourth phosphine herbicide marker gene of Bar in the plasmid pBinGlyBar1 selected in the present embodiment, therefore, careless fourth phosphine weedicide can be used T
1primary dcreening operation is carried out for butch flax seedling.Equally, if containing red fluorescent protein marker gene in the plasmid selected, then green Led light transmission red sunglasses sheet can be adopted to irradiate T
0for seed, discernable by eye goes out transgenosis T
0primary dcreening operation is carried out for seed.Select the vector plasmid carrying good riddled basins particularly important, experiment progress can be accelerated, shorten the screening cycle, effectively increase work efficiency.
(9) qualification of transfer-gen plant: cultivate T
1for plant, with the plant leaf of the primary dcreening operation positive for material, extract plant DNA, at expression vector G
2on to choose Bar gene fragment be template, utilize Vector NTI to design primer, design of primers result is as shown in table 1, adopts PCR method to identify primary dcreening operation positive plant, the results are shown in Figure 4.
Take DNA as template, with b-F and b-R sequence for primer, carry out pcr amplification, reaction system is 20 μ L, and concrete composition is in table 2.Response procedures: 95 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 2min, 35 circulations; 72 DEG C extend 10min.After reaction terminates, sample 3 μ L and adopt 1% agarose gel electrophoresis, detect whether amplify object fragment.
Claims (10)
1. an Agrobacterium tumefaciens mediated butch flax genetic transforming method, is characterized in that: described method is immersed in the agrobacterium tumefaciens bacterium liquid containing goal gene and screening-gene plasmid by the butch flax inflorescence come into bloom of pre-incubation, and concrete steps are as follows:
(1) the Construction and identification of engineering strain: adopt CaCl
2legal system for agrobacterium tumefaciens competent cell, average packing number pipe, be directly used in 24h conversion or liquid nitrogen flash freezer after-80 DEG C of preservations stand-by; Vector plasmid containing marker gene is imported in agrobacterium tumefaciens competent cell, often in pipe in agrobacterium tumefaciens competent cell: the volume ratio of YEB liquid nutrient medium is that the ratio of 1:8 adds YEB liquid nutrient medium, and mixing is placed on recovery 2h in 28 DEG C of environment; Centrifugal 45s under 4000 ~ 5000r/min room temperature, remove most of supernatant liquor, get the resuspended thalline of small part supernatant liquor, small part supernatant is 1:1 with the volume ratio of every pipe agrobacterium tumefaciens competent cell, is applied to by resuspended thalline on the YEB solid resistant panel substratum containing 50mg/L Rifampin and 50mg/L kantlex; Be inverted for 28 DEG C and cultivate 24-48h, YEB solid resistant panel substratum obtains resistant clones;
The positive list bacterium colony of the Agrobacterium tumefaciens strain of random picking fresh culture from YEB solid resistant panel substratum, be inoculated in the YEB liquid nutrient medium containing the Rifampin of 50mg/L and the kantlex of 50mg/L, 28 DEG C, 180r/min shaking culture is spent the night; Alkali cracking method extracts plasmid, and after agarose gel electrophoresis detects, enzyme cuts qualification recombinant plasmid, gets the single bacterium colony of the successful agrobacterium tumefaciens of qualification conversion stand-by;
(2) the preparation of butch flax seedling to be transformed: by vermiculite, Nutrition Soil, perlite mixing, this mixture will ensure the nutrition of ventilation property and soil, evenly sow butch flax seed, overlay film 2-3d is with slow seedling, remain on 20/16 DEG C of (day/night) natural lighting plantation to cultivate, when the inflorescence of butch flax plant less than 20% be in completely open, more than 60% be in period in bud time for transforming;
(3) the preparation of vacuum infiltration medium: picking qualification transforms the single colony inoculation of successful agrobacterium tumefaciens in the YEB liquid nutrient medium containing 50mg/L Rifampin, and 28 DEG C of overnight incubation obtain bacterium liquid; Bacterium liquid being transferred to volume is in the above-mentioned YEB liquid nutrient medium of bacterium liquid 100 times itself, 28 DEG C, 180r/min sustained oscillation cultivation 24-48h, the centrifugal 10min of 4000-6000r/min, abandon supernatant, with above-mentioned YEB liquid nutrient medium: the volume ratio of aseptic osmotic medium is the aseptic osmotic medium suspension thalline of the ratio of 1:40-60, mix, namely prepare the permeating medium of transformed plant, stand-by;
(4) contaminate and Dual culture: butch flax comes into bloom, and plant is put into a moisture eliminator higher than 300mm, connects a vacuum pump, butch flax inflorescence is inverted and is immersed in the moisture eliminator filling above-mentioned permeating medium, vacuum pump evacuation keeps 85kPa to transform 5min; Then, entangle butch flax inflorescence with freshness protection package, put Dual culture 24-48h under dark condition, remove bag, recover to cultivate 20/16 DEG C of (day/night) natural lighting, until it is ripe to plant pod, results T
0for seed;
(5) the primary dcreening operation of transgenic seed or plant: utilize marker gene contained by vector plasmid to butch flax T
0for seed or sowing T
0the T cultivated for seed
1primary dcreening operation is carried out for plant;
(6) the qualification of transfer-gen plant: cultivate T
1for plant, with the plant leaf of the primary dcreening operation positive for material, extract plant DNA, for marker gene or goal gene design primer, adopt round pcr qualification whether can amplify goal gene fragment, if identify successfully, namely obtain butch flax transfer-gen plant.
2. Agrobacterium tumefaciens mediated butch flax genetic transforming method according to claim 1, is characterized in that: described step (1) middle vector plasmid is the pBG1100 with kalamycin resistance, and this plasmid contains the anti-careless fourth phosphine herbicide marker gene of Bar.
3. Agrobacterium tumefaciens mediated butch flax genetic transforming method according to claim 2, is characterized in that: described step concrete steps (5), are (6) as follows:
(5) the primary dcreening operation of transgenic seed or plant: containing the anti-careless fourth phosphine herbicide marker gene of Bar in vector plasmid pBinGlyBar1, as plant strain growth 3cm long to two leaf phases, stem, be that the ratio of 1:300 is mixed to get mixing solutions with volume ratio by careless fourth phosphine weedicide and water, use this mixing solutions to spray T
1for butch flax seedling; Within spraying herbicide one week, most of plant is curling by blade, withered until whole strain is dead, only a few plant still can survive, and getting can the plant of survive, namely completes the preliminary screening to transfer-gen plant;
(6) the qualification of transfer-gen plant: cultivate T
1for plant, with the plant leaf of the primary dcreening operation positive for material, extract plant template DNA, carry out PCR qualification for marker gene Bar gene design primer sequence 1/ sequence 2, the object fragment of 420bp can be amplified in transgenic line, namely obtain butch flax transfer-gen plant.
4. Agrobacterium tumefaciens mediated butch flax genetic transforming method according to claim 1, is characterized in that: described step (2) middle vermiculite, Nutrition Soil, perlite is the mixing of 2:2:1 ratio in mass ratio;
Or described YEB solid resistant panel substratum consists of: 1.0g/L yeast extract paste+5.0g/L extractum carnis+5.0g/L peptone+0.5g/L MgSO
4+ 5.0g/L sucrose+15g/L agar+50mg/L Rifampin+50mg/L kantlex, pH7.0-7.4.
5. Agrobacterium tumefaciens mediated butch flax genetic transforming method according to claim 4, it is characterized in that: being prepared as follows of described YEB solid resistant panel substratum: after each component is mixed, pH is adjusted to 7.0-7.4,121 DEG C of sterilizing 20min, when substratum temperature is down to 60 DEG C, add Rifampin and kantlex again, it is stand-by that mixing is down flat plate.
6. Agrobacterium tumefaciens mediated butch flax genetic transforming method according to claim 1, it is characterized in that: described step (3) in aseptic osmotic medium consist of: the aqueous solution 5% (accounting for the mass percent of this substratum total mass) of 1/2MS substratum+sugar+efficient organic silicon surfactant 0.05% (accounting for the percent by volume of this substratum cumulative volume), pH 5.7.
7. the Agrobacterium tumefaciens mediated butch flax genetic transforming method according to claims 6, is characterized in that: in the aqueous solution of described sugar, sugar is glucose, fructose, semi-lactosi, sucrose or lactose.
8. Agrobacterium tumefaciens mediated butch flax genetic transforming method according to claim 6, it is characterized in that: being prepared as follows of described aseptic osmotic medium: the aqueous solution 5% (accounting for the mass percent of this substratum total mass) of 1/2MS substratum+sugar, pH is adjusted to 5.7, through 121 DEG C of sterilizing 20min, after temperature is down to normal temperature, add efficient organic silicon surfactant again, the nutrient solution after mixing is for the thalline that suspends.
9. Agrobacterium tumefaciens mediated butch flax genetic transforming method according to claim 1, it is characterized in that: described step concrete steps are (5) as follows: if containing DSRed red fluorescent protein marker gene in vector plasmid, then adopt green Led light transmission red sunglasses sheet to irradiate T
0for seed, what discernable by eye took on a red color fluorescence is transgenosis T
0for seed; If containing the anti-careless fourth phosphine herbicide marker gene of Bar in vector plasmid, then by T
1spray careless fourth phosphine weedicide for plant, can the preliminary judgement of normal growth be transfer-gen plant, carry out primary dcreening operation thus; Or, select other marker gene, the screening method that the different choice of the marker gene contained by vector plasmid is different.
10. the Agrobacterium tumefaciens mediated butch flax genetic transforming method according to any one of claim 1 to 9, is characterized in that: described agrobacterium tumefaciens is Agrobacterium tumefaciens strain EHA105.
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| CN111100871A (en) * | 2020-01-09 | 2020-05-05 | 天津科技大学 | Method for reducing browning and improving genetic transformation rate of agrobacterium-mediated castor |
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