CN103224954B - A kind of method of agriculture bacillus mediated cultivation genetically engineered soybean - Google Patents

A kind of method of agriculture bacillus mediated cultivation genetically engineered soybean Download PDF

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CN103224954B
CN103224954B CN201310193715.9A CN201310193715A CN103224954B CN 103224954 B CN103224954 B CN 103224954B CN 201310193715 A CN201310193715 A CN 201310193715A CN 103224954 B CN103224954 B CN 103224954B
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seedling
agrobacterium
suspension
soybean
dual culture
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CN103224954A (en
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陈李淼
沙爱华
张晓娟
单志慧
陈水莲
张婵娟
邱徳珍
周蓉
周新安
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Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
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Abstract

The invention discloses a kind of method of agriculture bacillus mediated cultivation genetically engineered soybean.The method is, by agriculture bacillus mediated, target DNA is proceeded to soya cells, comprises the steps: 1) after the suspension of the recombinational agrobacterium of the cotyledonary node meristematic tissue place of Germination of Soybean Seed seedling injection containing target DNA, described seedling is carried out Dual culture; 2) the described seedling through step 1) Dual culture is directly carried out long seedling cultivation, namely obtain the Transgenic soybean plants with root, stem and leaf.The present invention have simple to operate, the transformation period is short, the advantage of transformation efficiency high (reaching as high as 15.3%), economy, applied range.

Description

A kind of method of agriculture bacillus mediated cultivation genetically engineered soybean
Technical field
The present invention relates to a kind of method of agriculture bacillus mediated cultivation genetically engineered soybean.
Background technology
Compared with traditional breeding technique, the cycle of transgenic breeding technology is short, efficiency is high, purpose is strong, is the effective way of Crop Improvement economical character.At present, soybean gene-transferring method mainly contains agrobacterium-mediated transformation, particle bombardment, pollen tube passage method, and wherein conventional is agrobacterium-mediated transformation, and T-DNA can more intactly insert in Plant Genome with single copy or low copy form by the method.Although in recent years, the technology of Agrobacterium-mediated transformation soybean was constantly perfect, and most method through the dedifferentiation of explant and the process of breaking up again, and need exist the problems such as transformation efficiency is low, genetic stability is poor, the transformation period is long.These problems seriously hinder the checking of functional gene and the process of transgenic breeding.
Summary of the invention
The object of this invention is to provide a kind of method of cultivating genetically engineered soybean, target DNA is proceeded to soya cells by agriculture bacillus mediated by the method, comprises the steps:
1) after the suspension of the recombinational agrobacterium of the cotyledonary node meristematic tissue place of Germination of Soybean Seed seedling injection containing target DNA, described seedling is carried out Dual culture;
2) the described seedling through step 1) Dual culture is directly carried out long seedling cultivation, namely obtain the Transgenic soybean plants with root, stem and leaf;
The object of described Dual culture is that described target DNA is incorporated in soybean gene group.
In the above-mentioned methods, described seedling during described injection is the seedling of seed germination 5-10 days (as 5,7 or 10 days).
In the above-mentioned methods, because the terminal bud on described seedling and lateral bud can develop into new tissue, in order to avoid the generation of non-genetically modified organism, make the transgenic trait that whole plant is consistent, before described injection, also can comprise the step terminal bud of described seedling and lateral bud removed.
In the above-mentioned methods, the suspension of described recombinational agrobacterium is according to the method preparation comprised the steps: described recombinational agrobacterium is suspended in the suspension forming described recombinational agrobacterium in the liquid nutrient medium with following composition: solvent is water, solute and the final concentration in described liquid nutrient medium thereof are respectively: the B5 minimum medium inorganic salts ingredients of 1/10, B5 minimum medium organic composition, sucrose 30g/L, 2-(N-morpholine) ethyl sulfonic acid 3.9g/L, 6-benzyladenine 1.67mg/L, Cys 400-1000mg/L(is as 400, 700, or 1000mg/L), dithiothreitol (DTT) 154mg/L, Sulfothiorine 158mg/L, Syringylethanone 0-300 μm ol/L(or be 50-300 μm of ol/L, 100-300 μm of ol/L, 150-300 μm of ol/L or 200-300 μm of ol/L, specifically can be 0 μm of ol/L, 50 μm of ol/L, 100 μm of ol/L, 150 μm of ol/L, 200 μm of ol/L, or 300 μm of ol/L), Plant hormones regulators,gibberellins 0.1mg/L,
The B5 minimum medium inorganic salts ingredients of described 1/10 is KNO 3250mg/L, CaCl 2113mg/L, MgSO 4122mg/L, (NH 4) 2sO 413.4mg/L, KI0.075mg/L, H 3bO 40.3mg/L, MnSO 47mg/L, ZnSO 41.15mg/L, Na 2moO 40.21mg/L, CoCl 20.014mg/L, CuSO 40.016mg/L, Na 2-EDTA3.73mg/L, FeSO 415.2mg/L;
Described B5 minimum medium organic composition is inositol 100mg/L, nicotinic acid 1mg/L, pyridoxine hydrochloride 1mg/L, Tyiamine Hd 10mg/L.
In the above-mentioned methods, compared with described liquid nutrient medium, the OD of the suspension of described recombinational agrobacterium 600value is that 0.4-1.0(is as 0.4,0.6,1.0); The injection volume of the suspension of described recombinational agrobacterium is each described seedling 20-100 μ L (as 20,80,100 μ L).
In the above-mentioned methods, be injected through described in internal diameter be 0.06mm-1mm (as 0.06,0.1 or 1mm), external diameter is 0.2mm-1.14mm(as 0.2,0.24,1.14mm) syringe needle realize.
In the above-mentioned methods, described Agrobacterium is agrobacterium tumefaciens, specifically can be agrobacterium tumefaciens EHA105.
In the above-mentioned methods, the condition of described Dual culture is use Aquaponic 3 days under temperature 25 DEG C, dark condition.
Present invention also offers the liquid nutrient medium that a kind of Agrobacterium infects plant, its solvent is water, solute and the final concentration in described liquid nutrient medium thereof are respectively: the B5 minimum medium inorganic salts ingredients of 1/10, B5 minimum medium organic composition, sucrose 30g/L, 2-(N-morpholine) ethyl sulfonic acid 3.9g/L, 6-benzyladenine 1.67mg/L, Cys 400-1000mg/L, dithiothreitol (DTT) 154mg/L, Sulfothiorine 158mg/L, Syringylethanone 0-400 μm ol/L(or be 0-300 μm of ol/L, 50-300 μm of ol/L, 100-300 μm of ol/L, 150-300 μm of ol/L or 200-300 μm of ol/L, specifically can be 0 μm of ol/L, 50 μm of ol/L, 100 μm of ol/L, 150 μm of ol/L, 200 μm of ol/L, or 300 μm of ol/L), Plant hormones regulators,gibberellins 0.1mg/L,
The B5 minimum medium inorganic salts ingredients of described 1/10 is KNO 3250mg/L, CaCl 2113mg/L, MgSO 4122mg/L, (NH 4) 2sO 413.4mg/L, KI0.075mg/L, H 3bO 40.3mg/L, MnSO 47mg/L, ZnSO 41.15mg/L, Na 2moO 40.21mg/L, CoCl 20.014mg/L, CuSO 40.016mg/L, Na 2-EDTA3.73mg/L, FeSO 415.2mg/L;
Described B5 minimum medium organic composition is inositol 100mg/L, nicotinic acid 1mg/L, pyridoxine hydrochloride 1mg/L, Tyiamine Hd 10mg/L.
In above-mentioned substratum, described plant is monocotyledons or dicotyledons, and described dicotyledons specifically can be soybean.
There is according to plant meristematic tissue the characteristic of mitogenetic new cell, and for producing and break up the basis of other various tissues, the present invention adopts the mode sentencing injection agrobacterium suspension at the cotyledonary node meristematic tissue of Germination of Soybean Seed seedling, target DNA is integrated into the karyomit(e) of soybean, obtain transfer-gen plant, whole process is without the dedifferentiation of plant tissue and the tissue culture procedures of breaking up again, not by experiment condition (super clean benches such as aseptic techniques, group training between) restriction, decrease chemical reagent (as hormone, fungistat) usage quantity, have simple to operate, transformation period is short, transformation efficiency high (reaching as high as 15.3%), economy, the advantage of applied range.
Accompanying drawing explanation
Fig. 1 is that soybean seedling cotyledonary node meristematic tissue figure is infected in injection.
Fig. 2 is that blade smears the plant of careless ammonium phosphine after 3 days.
Fig. 3 is the result that PCR detects herbicide resistance gene Bar in transfer-gen plant.Wherein, swimming lane M is molecular weight standard, and size is from top to bottom followed successively by 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp, and swimming lane 1-24 is the amplified production of transfer-gen plant to be measured.
Fig. 4 uses different concns Syringylethanone on the impact of transformation efficiency in restructuring agrobacterium suspension.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Embodiment 1, agriculture bacillus mediated cultivation genetically engineered soybean
Scheme I
1, the preparation of recombinational agrobacterium suspension
Plant binary expression vector PB2GW7(Binary gatewayoverexpression vector PB2GW7 by containing herbicide resistance gene Bar) (document: Dubin MJ, Bowler C, Benvenuto G.A modifiedGateway cloning strategy for overexpressing tagged proteins in plants.Plantmethods, 2008, 4 (3): the 1-11. public can obtain from Inst. of Oil Crops, Chinese Academy of Agriculture) proceed to agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105(purchased from sky, Beijing bounties Gene Tech. Company Limited), obtain the restructuring agrobacterium tumefaciens EHA105/PB2GW7 containing goal gene.
Single bacterium colony of restructuring agrobacterium tumefaciens EHA105/PB2GW7 is cultured to OD with the YEP nutrient solution (solvent is water, and solute and concentration thereof are respectively: sodium-chlor 5g/L, yeast extract 5g/L and Tryptones 10g/L) containing spectinomycin 50mg/L and Rifampin 25mg/L in 28 DEG C 600be 0.8-1.2; 4000rpm, centrifugal 10min, resuspended to OD with following liquid nutrient medium 600be 0.6 suspension obtaining recombinational agrobacterium:
Solvent is water, and solute and the final concentration in described liquid nutrient medium thereof are respectively: the B5 minimum medium inorganic salts ingredients of 1/10, B5 minimum medium organic composition, sucrose 30g/L, 2-(N-morpholine) ethyl sulfonic acid 3.9g/L, 6-benzyladenine 1.67mg/L, Cys 1000mg/L, dithiothreitol (DTT) 154mg/L, Sulfothiorine 158mg/L, Syringylethanone 200 μm of ol/L, Plant hormones regulators,gibberellins 0.1mg/L;
The B5 minimum medium inorganic salts ingredients of described 1/10 is KNO 3250mg/L, CaCl 2113mg/L, MgSO 4122mg/L, (NH 4) 2sO 413.4mg/L, KI0.075mg/L, H 3bO 40.3mg/L, MnSO 47mg/L, ZnSO 41.15mg/L, Na 2moO 40.21mg/L, CoCl 20.014mg/L, CuSO 40.016mg/L, Na 2-EDTA3.73mg/L, FeSO 415.2mg/L;
Described B5 minimum medium organic composition is inositol 100mg/L, nicotinic acid 1mg/L, pyridoxine hydrochloride 1mg/L, Tyiamine Hd 10mg/L.
2, the preparation of seedling
Full and the surperficial soybean varieties without scab east agriculture 50 seed of picking, keep at a certain distance away and be placed between wetting double-layer filter paper, parcel in paper roll, is put in water planting in plastics casing, 25 DEG C, the photoperiod be illumination every day 16 hours, dark 8 hours, intensity of illumination is 100 μm of olm -2s -1condition under be cultured to seed germination 7 days, remove terminal bud and lateral bud, the injection carrying out step 3 is infected.
3, injection is infected and Dual culture
Get the suspension of recombinational agrobacterium prepared by step 1, by internal diameter be 0.1mm, external diameter is the cotyledonary node meristematic tissue place of tubulose needle injection (Fig. 1) to the described young shoot of the removal terminal bud of step 2 and lateral bud of 0.24mm, each described seedling injects 80 μ L, described seedling is placed in 25 DEG C, dark, humidity is by Aquaponic (i.e. Dual culture) 3 days under the condition of 70%.
4, seedling is cultivated and screening
Seedling replanting through step 3 Dual culture is cultivated about 10 days to soil under normal water and fertilizer condition, until after growing ternately compound leaf, ternated compound blade is smeared with the glufosinates of 100mg/L, after 3 days, observe blade whether to turn yellow, as shown in Figure 2, the negative plant of wild-type non-genetically engineered soybean east agriculture 20 smears blade flavescence (the left figure in Fig. 2) of glufosinates to partial results, and the blade that transfer-gen plant smears glufosinates is still green (the right figure in Fig. 2).Remove the plant that blade turns yellow, retain the plant of blade without considerable change.
5, PCR qualification
Step 4 of learning from else's experience is through the plant of glufosinates screening without considerable change, extract genomic dna, with this DNA for template carries out the pcr amplification of resistant gene Bar, amplified production is carried out 1% agarose gel electrophoresis, the plant of the Bar gene fragment containing 440bp in amplified production is positive transgenic plant, plant not containing this fragment is negative transfer-gen plant, and part amplification as shown in Figure 3.
The primer sequence that above-mentioned pcr amplification uses is as follows:
The forward primer of amplification Bar gene: 5'-GCACCATCGTCAACCACTAC-3';
The reverse primer of amplification Bar gene: 5'-TGAAGTCCAGCTGCCAGAAAC-3'.
6, experimental result
The Setup Experiments of step 1-5 repeats for three times, and result is as shown in table 1.
The statistics of table 1, agriculture bacillus mediated cultivation genetically engineered soybean
Note: transformation efficiency=(PCR identifies positive plant number/injection seedling number) × 100%
Scheme II
1, the preparation of recombinational agrobacterium suspension
Carry out according to the method for step 1 in scheme I, difference is: the final concentration of the Cys in the liquid nutrient medium that the restructuring agrobacterium tumefaciens that suspends uses is 700mg/L; Relative to described liquid nutrient medium, the OD of recombinational agrobacterium suspension 600be 0.4.
2, the preparation of seedling
Carry out according to the method for step 2 in scheme I, difference is: be the seed germination seedling of 5 days for injecting the seedling of infecting.
3, injection is infected and Dual culture
Get the suspension of recombinational agrobacterium prepared by step 1, by internal diameter be 0.06mm, external diameter is the cotyledonary node meristematic tissue place that the tubulose syringe needle of 0.20mm is expelled to the removal terminal bud of step 2 and the described young shoot of lateral bud, each described seedling injects 100 μ L, described seedling is placed in 25 DEG C, dark, humidity is by Aquaponic (i.e. Dual culture) 3 days under the condition of 70%.
4, seedling is cultivated and screening
Carry out according to the method for step 4 in scheme I.
5, PCR qualification
Carry out according to the method for step 5 in scheme I.
6, result
With the result of scheme I without significant difference.
Scheme III
1, the preparation of recombinational agrobacterium suspension
Carry out according to the method for step 1 in scheme I, difference is: the final concentration of the Cys in the liquid nutrient medium that the restructuring agrobacterium tumefaciens that suspends uses is 400mg/L; Relative to described liquid nutrient medium, the OD of recombinational agrobacterium suspension 600be 1.0.
2, the preparation of seedling
Carry out according to the method for step 2 in scheme I, difference is: be the seed germination seedling of 10 days for injecting the seedling of infecting.
3, injection is infected and Dual culture
Get the suspension of recombinational agrobacterium prepared by step 1, by internal diameter be 1mm, external diameter is the cotyledonary node meristematic tissue place that the tubulose syringe needle of 1.14mm is expelled to the removal terminal bud of step 2 and the described young shoot of lateral bud, each described seedling injects 20 μ L, described seedling is placed in 25 DEG C, dark, humidity is by Aquaponic (i.e. Dual culture) 3 days under the condition of 70%.
4, seedling is cultivated and screening
Carry out according to the method for step 4 in scheme I.
5, PCR qualification
Carry out according to the method for step 5 in scheme I.
6, result
With the result of scheme I without significant difference.
In embodiment 2, agrobacterium suspension, Syringylethanone concentration is on the impact of transformation efficiency
1, the preparation of recombinational agrobacterium suspension
By expression vector pCambia3301(purchased from Beijing DingGuo ChangSheng Biology Technology Co., Ltd, containing gus reporter gene) proceed to agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105(purchased from sky, Beijing bounties Gene Tech. Company Limited) in, obtain the restructuring agrobacterium tumefaciens EHA105/pCambia3301 containing gus reporter gene.
Single bacterium colony of restructuring agrobacterium tumefaciens EHA105/pCambia3301 is cultured to OD with the YEP nutrient solution (solvent is water, and solute and concentration thereof are respectively: sodium-chlor 5g/L, yeast extract 5g/L and Tryptones 10g/L) containing spectinomycin 50mg/L and Rifampin 25mg/L in 28 DEG C 600be 0.8-1.2; 4000rpm, centrifugal 10min, resuspended to OD with following liquid nutrient medium 600be 0.6 suspension obtaining recombinational agrobacterium:
Solvent is water, and solute and the final concentration in described liquid nutrient medium thereof are respectively: the B5 minimum medium inorganic salts ingredients of 1/10, B5 minimum medium organic composition, sucrose 30g/L, 2-(N-morpholine) ethyl sulfonic acid 3.9g/L, 6-benzyladenine 1.67mg/L, Cys 1000mg/L, dithiothreitol (DTT) 154mg/L, Sulfothiorine 158mg/L, syringylethanone 0,50,100,150,200 or 300 μm of ol/L, Plant hormones regulators,gibberellins 0.1mg/L;
The B5 minimum medium inorganic salts ingredients of described 1/10 is KNO 3250mg/L, CaCl 2113mg/L, MgSO 4122mg/L, (NH 4) 2sO 413.4mg/L, KI0.075mg/L, H 3bO 40.3mg/L, MnSO 47mg/L, ZnSO 41.15mg/L, Na 2moO 40.21mg/L, CoCl 20.014mg/L, CuSO 40.016mg/L, Na 2-EDTA3.73mg/L, FeSO 415.2mg/L;
Described B5 minimum medium organic composition is inositol 100mg/L, nicotinic acid 1mg/L, pyridoxine hydrochloride 1mg/L, Tyiamine Hd 10mg/L.
2, the preparation of seedling
Carry out according to the method for step 2 in embodiment 1 scheme I.
3, injection is infected and Dual culture
Carry out according to the method for step 3 in embodiment 1 scheme I.
4, GUS staining examine
By the seedling through step 3 Dual culture, get cotyledonary node position and carry out GUS dyeing, statistics transformation efficiency (%), namely cotyledonary node position becomes the per-cent that blue seedling number accounts for the plant sum infected through step 1 same concentrations Syringylethanone (AS) recombinational agrobacterium suspension, and result is as shown in table 1 with shown in Fig. 4.
The method of above-mentioned GUS dyeing is as follows:
By cotyledonary node 90% acetone treatment 15min, add GUS dye liquor 37 DEG C process 16h after 0.1M phosphate buffered saline buffer (PBS) washing, after ethanol decolorization process, entity microscope observing, with not genetically modified WT lines for contrast.GUS dye liquor forms: 0.5mg/ml X-Glu, 10mM phosphate buffered saline buffer (PBS), the 0.5mM Tripotassium iron hexacyanide, 0.5mM yellow prussiate of potash, 0.1%Triton X-100,10mM EDTA.
Table 1, gus gene transient expression Efficiency testing result
AS concentration (μm ol/L) 0 50 100 150 200 300
Transformation efficiency (%) 10.6b 25.2b 50.0c 63.4a 72.0a 33.6b
Note: in table, data are 3 mean values repeated; Be marked with different lowercase person difference after same column result and reach 5% conspicuous level.
Embodiment 3, different soybean varieties are on the impact of transformation efficiency
Select eastern agriculture 50, middle yellow 35, Shanxi beans 21, middle beans 33, middle beans 40, grand No. one of sky respectively, cultivate rich 16 and 48 8, Heihe soybean varieties carry out transformation efficiency analysis.Concrete grammar is as follows:
1, the preparation of recombinational agrobacterium suspension
By expression vector pCambia3301(purchased from Beijing DingGuo ChangSheng Biology Technology Co., Ltd, containing herbicide resistance gene Bar) proceed to agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105(purchased from sky, Beijing bounties Gene Tech. Company Limited) in, obtain the restructuring agrobacterium tumefaciens EHA105/pCambia3301 containing gus reporter gene.
Single bacterium colony of restructuring agrobacterium tumefaciens EHA105/pCambia3301 is cultured to OD with the YEP nutrient solution (solvent is water, and solute and concentration thereof are respectively: sodium-chlor 5g/L, yeast extract 5g/L and Tryptones 10g/L) containing spectinomycin 50mg/L and Rifampin 25mg/L in 28 DEG C 600be 0.8-1.2; 4000rpm, centrifugal 10min, resuspended to OD with following liquid nutrient medium 600be 0.6 suspension obtaining recombinational agrobacterium:
Solvent is water, and solute and the final concentration in described liquid nutrient medium thereof are respectively: the B5 minimum medium inorganic salts ingredients of 1/10, B5 minimum medium organic composition, sucrose 30g/L, 2-(N-morpholine) ethyl sulfonic acid 3.9g/L, 6-benzyladenine 1.67mg/L, Cys 1000mg/L, dithiothreitol (DTT) 154mg/L, Sulfothiorine 158mg/L, Syringylethanone 200 μm of ol/L, Plant hormones regulators,gibberellins 0.1mg/L;
The B5 minimum medium inorganic salts ingredients of described 1/10 is KNO 3250mg/L, CaCl 2113mg/L, MgSO 4122mg/L, (NH 4) 2sO 413.4mg/L, KI0.075mg/L, H 3bO 40.3mg/L, MnSO 47mg/L, ZnSO 41.15mg/L, Na 2moO 40.21mg/L, CoCl 20.014mg/L, CuSO 40.016mg/L, Na 2-EDTA3.73mg/L, FeSO 415.2mg/L;
Described B5 minimum medium organic composition is inositol 100mg/L, nicotinic acid 1mg/L, pyridoxine hydrochloride 1mg/L, Tyiamine Hd 10mg/L.
2, the preparation of seedling
Full and the surperficial soybean varieties without the scab east agriculture 50 of picking, middle yellow 35, Shanxi beans 21, middle beans 33, middle beans 40, grand No. one of sky, cultivate rich 16 or Heihe 48 seed, keep at a certain distance away and be placed between wetting double-layer filter paper, parcel is in paper roll, be put in water planting in plastics casing, 25 DEG C, the photoperiod be illumination every day 16 hours, dark 8 hours, intensity of illumination is 100 μm of olm -2s -1condition under be cultured to seed germination 7 days, remove terminal bud and lateral bud, the injection carrying out step 3 is infected.
3, injection is infected and Dual culture
Carry out according to the method for step 3 in embodiment 1 scheme I.
4, seedling is cultivated and screening
Carry out according to the method for step 4 in scheme I.
5, PCR qualification
Step 4 of learning from else's experience is through the plant of glufosinates screening without considerable change, extract genomic dna, with this DNA for template carries out the pcr amplification of resistant gene Bar, amplified production is carried out 1% agarose gel electrophoresis, the plant of the Bar gene fragment containing 440bp in amplified production is positive transgenic plant, and the plant not containing this fragment is negative transfer-gen plant.
The primer sequence that above-mentioned pcr amplification uses is as follows:
The forward primer of amplification Bar gene: 5'-GCACCATCGTCAACCACTAC-3';
The reverse primer of amplification Bar gene: 5'-TGAAGTCCAGCTGCCAGAAAC-3'.
6, result: as shown in table 3.
The transformation efficiency statistics of table 3, different soybean varieties
Note: transformation efficiency=(PCR identifies positive plant number/injection seedling number) × 100%

Claims (4)

1. cultivate the method for genetically engineered soybean, by agriculture bacillus mediated, target DNA is proceeded to soya cells, it is characterized in that: described method comprises the steps:
1) within Germination of Soybean Seed 5-10 days, obtain seedling, before injection, seedling terminal bud and lateral bud are removed, then after the suspension of the recombinational agrobacterium of its cotyledonary node meristematic tissue place injection containing target DNA, described seedling is carried out Dual culture;
2) by through step 1) the described seedling of Dual culture directly carries out long seedling cultivation, namely obtains the Transgenic soybean plants with root, stem and leaf;
The suspension of described recombinational agrobacterium is according to the method preparation comprised the steps: described recombinational agrobacterium is suspended in the suspension forming described recombinational agrobacterium in the liquid nutrient medium with following composition: solvent is water, solute and the final concentration in described liquid nutrient medium thereof are respectively: B5 minimum medium organic composition, the B5 minimum medium inorganic salts ingredients of 1/10, sucrose 30g/L, 2-(N-morpholine) ethyl sulfonic acid 3.9g/L, 6-benzyladenine 1.67mg/L, Cys 400-1000mg/L, dithiothreitol (DTT) 154mg/L, Sulfothiorine 158mg/L, Syringylethanone 0-300 μm ol/L, Plant hormones regulators,gibberellins 0.1mg/L,
Described B5 minimum medium organic composition is inositol 100mg/L, nicotinic acid 1mg/L, pyridoxine hydrochloride 1mg/L, Tyiamine Hd 10mg/L;
The B5 minimum medium inorganic salts ingredients of described 1/10 is KNO 3250mg/L, CaCl 2113mg/L, MgSO 4122mg/L, (NH 4) 2sO 413.4mg/L, KI 0.075mg/L, H 3bO 40.3mg/L, MnSO 47mg/L, ZnSO 41.15mg/L, Na 2moO 40.21mg/L, CoCl 20.014mg/L, CuSO 40.016mg/L, Na 2-EDTA 3.73mg/L, FeSO 415.2mg/L;
Compared with described liquid nutrient medium, the OD of the suspension of described recombinational agrobacterium 600value is 0.4-1.0;
The injection volume of the suspension of described recombinational agrobacterium is each described seedling 20-100 μ L;
Describedly be injected through internal diameter is 0.06mm-1mm, external diameter is 0.20mm-1.14mm syringe needle and realize.
2. method according to claim 1, is characterized in that: described Agrobacterium is agrobacterium tumefaciens.
3. method according to claim 2, is characterized in that: described Agrobacterium is agrobacterium tumefaciens EHA105.
4. method according to claim 1 and 2, is characterized in that: the condition of described Dual culture for using Aquaponic 3 days under temperature 25 DEG C, dark condition.
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