CN103224954A - Method for breeding transgenic soybeans through Agrobacterium tumefaciens mediated transformation - Google Patents

Abstract

The invention discloses a method for breeding transgenic soybeans through Agrobacterium tumefaciens mediated transformation. The method is characterized by transferring a target DNA (deoxyribonucleic acid) into soybean cells through Agrobacterium tumefaciens mediated transformation, and comprises the following steps: 1) injecting a suspension of recombinant Agrobacterium tumefaciens containing the target DNA at cotyledonary node meristematic tissues of soybean seeds where seedlings are germinated, and then co-culturing the seedlings; and 2) directly performing long seedling culture on the seedlings co-cultured in the step 1) to obtain transgenic soybean plants having roots, stems and leaves. The invention has the advantages of simple operation process, short transformation period, high transformation efficiency (the maximum can be up to 15.3%), high economy and wide application 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

Compare with traditional breeding technique, the cycle of transgenic breeding technology is short, efficient is high, purpose is strong, is the effective way of improvement crop economical character.At present, the soybean transgene method mainly contains agrobacterium-mediated transformation, particle bombardment, pollen tube passage method, and wherein commonly used is agrobacterium-mediated transformation, and this method can more intactly be inserted T-DNA in the Plant Genome with single copy or low copy form.Though in recent years, the technology of agrobacterium-mediated transformation soybean transformation was constantly perfect, and most methods need be through the dedifferentiation of explant and process of differentiation again, and has problems such as transformation efficiency is low, genetic stability is poor, the transformation period is long.These problems have seriously hindered the checking of functional gene and the process of transgenic breeding.

Summary of the invention

The purpose of this invention is to provide a kind of method of cultivating genetically engineered soybean, this method changes target DNA over to soya cells by agriculture bacillus mediated, comprises the steps:

1) after the suspension of the reorganization Agrobacterium that contains target DNA is injected at the cotyledonary node meristematic tissue place of soybean seeds sprouting seedling, described seedling is carried out common cultivation;

2) will cultivate through the directly long seedling of the described seedling that step 1) is cultivated altogether, promptly obtain having the genetically engineered soybean plant of root, stem and leaf;

Described purpose of cultivating altogether is that described target DNA is incorporated in the soybean gene group.

In aforesaid method, the described seedling during described the injection is the seedling of seed germination 5-10 days (as 5,7 or 10 days).

In aforesaid method, because terminal bud and lateral bud on the described seedling can develop into new tissue, for fear of the not generation of genetically modified organism, the transgenosis characteristic that whole plant is consistent, before described injection, also can comprise the step that terminal bud and the lateral bud of described seedling are removed.

In aforesaid method, the suspension of described reorganization Agrobacterium prepares according to the method that comprises the steps: described reorganization Agrobacterium is suspended in the suspension that forms described reorganization Agrobacterium in the liquid nutrient medium with following composition: solvent is a water, and solute and the final concentration in described liquid nutrient medium thereof are respectively: 1/10 B5 minimum medium inorganic salt composition, B5 minimum medium organic composition, sucrose 30g/L, 2-(N-morpholine) ethyl sulfonic acid 3.9g/L, 6-benzyladenine 1.67mg/L, L-halfcystine 400-1000mg/L(is as 400,700, or 1000mg/L), dithiothreitol (DTT) 154mg/L, Sulfothiorine 158mg/L, Syringylethanone 0-300 μ mol/L(or be 50-300 μ mol/L, 100-300 μ mol/L, 150-300 μ mol/L or 200-300 μ mol/L, specifically can be 0 μ mol/L, 50 μ mol/L, 100 μ mol/L, 150 μ mol/L, 200 μ mol/L, or 300 μ mol/L), Plant hormones regulators,gibberellins 0.1mg/L;

Described 1/10 B5 minimum medium inorganic salt composition 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, hydrochloric acid sulphur ammonium 10mg/L.

In aforesaid method, compare the OD of the suspension of described reorganization Agrobacterium with described liquid nutrient medium ₆₀₀Value is that 0.4-1.0(is as 0.4,0.6,1.0); The injection volume of the suspension of described reorganization Agrobacterium is each described seedling 20-100 μ L (as 20,80,100 μ L).

In aforesaid method, described injection by 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 aforesaid method, described Agrobacterium is an agrobacterium tumefaciens, specifically can be agrobacterium tumefaciens EHA105.

In aforesaid method, described culture condition altogether is that water was cultivated 3 days under 25 ℃ of temperature, dark condition.

The present invention also provides a kind of Agrobacterium to infect the liquid nutrient medium that plant is used, its solvent is a water, and solute and the final concentration in described liquid nutrient medium thereof are respectively: 1/10 B5 minimum medium inorganic salt composition, B5 minimum medium organic composition, sucrose 30g/L, 2-(N-morpholine) ethyl sulfonic acid 3.9g/L, 6-benzyladenine 1.67mg/L, L-halfcystine 400-1000mg/L, dithiothreitol (DTT) 154mg/L, Sulfothiorine 158mg/L, Syringylethanone 0-400 μ mol/L(or be 0-300 μ mol/L, 50-300 μ mol/L, 100-300 μ mol/L, 150-300 μ mol/L or 200-300 μ mol/L, specifically can be 0 μ mol/L, 50 μ mol/L, 100 μ mol/L, 150 μ mol/L, 200 μ mol/L, or 300 μ mol/L), Plant hormones regulators,gibberellins 0.1mg/L;

Described 1/10 B5 minimum medium inorganic salt composition 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, hydrochloric acid sulphur ammonium 10mg/L.

In above-mentioned substratum, described plant is monocotyledons or dicotyledons, and described dicotyledons specifically can be soybean.

The characteristic that has mitogenetic new cell according to the plant meristematic tissue, and for producing and break up the basis of other various tissues, the present invention adopts the mode of sentencing the injection agrobacterium suspension at the cotyledonary node meristematic tissue of soybean seeds sprouting 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 differentiated tissues culturing process again, be not subjected to experiment condition (super clean benches such as aseptic technique, between group training) restriction, reduced chemical reagent (as hormone, fungistat) usage quantity has simple to operate, transformation period is short, transformation efficiency height (reaching as high as 15.3%), economy, the advantage of applied range.

Description of drawings

Fig. 1 infects soybean seedling cotyledonary node meristematic tissue figure for injection.

Fig. 2 smears the plant of careless ammonium phosphine after 3 days for blade.

Fig. 3 detects the result of herbicide resistance gene Bar in the transfer-gen plant for PCR.Wherein, swimming lane M is a molecular weight standard, and size from top to bottom is 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 is for using the influence of different concns Syringylethanone to transformation efficiency in the reorganization agrobacterium suspension.

Embodiment

Employed experimental technique is ordinary method if no special instructions among the following embodiment.

Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.

Embodiment 1, agriculture bacillus mediated cultivation genetically engineered soybean

The scheme I

1, the preparation of reorganization agrobacterium suspension

The plant binary expression vector PB2GW7(Binary gateway overexpression vector PB2GW7 that will contain herbicide resistance gene Bar) (document: Dubin MJ, Bowler C, Benvenuto G.A modified Gateway cloning strategy for overexpressing tagged proteins in plants.Plant methods, 2008,4 (3): the 1-11. public can obtain from Inst. of Oil Crops, Chinese Academy of Agriculture) change agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105(over to available from sky, Beijing bounties Gene Tech. Company Limited), obtain containing the reorganization agrobacterium tumefaciens EHA105/PB2GW7 of goal gene.

Single bacterium colony of reorganization agrobacterium tumefaciens EHA105/PB2GW7 is cultured to OD with the YEP nutrient solution (solvent is a water, and solute and concentration thereof are respectively: sodium-chlor 5g/L, yeast extract 5g/L and Tryptones 10g/L) that contains spectinomycin 50mg/L and Rifampin 25mg/L in 28 ℃ ₆₀₀Be 0.8-1.2; 4000rpm, centrifugal 10min, resuspended with following liquid nutrient medium to OD ₆₀₀Be 0.6 suspension that obtains recombinating Agrobacterium:

Solvent is a water, and solute and the final concentration in described liquid nutrient medium thereof are respectively: 1/10 B5 minimum medium inorganic salt composition, B5 minimum medium organic composition, sucrose 30g/L, 2-(N-morpholine) ethyl sulfonic acid 3.9g/L, 6-benzyladenine 1.67mg/L, L-halfcystine 1000mg/L, dithiothreitol (DTT) 154mg/L, Sulfothiorine 158mg/L, Syringylethanone 200 μ mol/L, Plant hormones regulators,gibberellins 0.1mg/L;

Described 1/10 B5 minimum medium inorganic salt composition 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, hydrochloric acid sulphur ammonium 10mg/L.

2, the preparation of seedling

Eastern agricultural 50 seeds of picking soybean varieties full and surperficial no scab, keep at a certain distance away and place between wetting double-deck filter paper, parcel is paper roll, is put in water planting in the plastics casing, and 25 ℃, photoperiod are that illumination every day 16 hours, dark 8 hours, intensity of illumination are 100 μ molm ^-2S ^-1Condition under be cultured to seed germination 7 days, remove terminal bud and lateral bud, carry out the injection of step 3 and infect.

3, injection is infected and is cultivated altogether

Get the suspension of the reorganization Agrobacterium of step 1 preparation, by internal diameter is that 0.1mm, external diameter are the cotyledonary node meristematic tissue place of the tubulose needle injection (Fig. 1) of 0.24mm to the described young shoot of the removal terminal bud of step 2 and lateral bud, each described seedling is injected 80 μ L, and it is that water was cultivated (promptly cultivating altogether) 3 days under 70% the condition that described seedling is placed 25 ℃, dark, humidity.

4, become seedling to cultivate and screening

To soil, cultivate about 10 days down through the seedling replanting that step 3 is cultivated altogether in normal water and fertilizer condition, after growing ternately compound leaf, smear ternated compound blade with the glufosinates of 100mg/L, after 3 days, observe whether flavescence of blade, partial results as shown in Figure 2, the not negative plant of the genetically engineered soybean east farming 20 blade flavescence (the left figure among Fig. 2) of smearing glufosinates of wild-type, the blade that transfer-gen plant is smeared glufosinates still is green (the right figure among Fig. 2).Remove the plant of blade flavescence, keep the plant that blade does not have considerable change.

5, PCR identifies

The step 4 of learning from else's experience is through the plant of the no considerable change of glufosinates screening, extract genomic dna, with this DNA is the pcr amplification that template is carried out resistant gene Bar, amplified production is carried out 1% agarose gel electrophoresis, the positive transfer-gen plant of plant that contains the Bar gene fragment of 440bp in the amplified production, do not contain the negative transfer-gen plant of this segmental plant, the 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 experiment of step 1-5 is provided with three repetitions, and the result is as shown in table 1.

The statistics of table 1, agriculture bacillus mediated cultivation genetically engineered soybean

Annotate: transformation efficiency=(PCR identifies positive plant number/injection seedling number) * 100%

The scheme II

1, the preparation of reorganization agrobacterium suspension

Method according to step 1 in the scheme I is carried out, and difference is: the final concentration of the L-halfcystine in the employed liquid nutrient medium of reorganization agrobacterium tumefaciens that suspends is 700mg/L; With respect to described liquid nutrient medium, the OD of reorganization agrobacterium suspension ₆₀₀Be 0.4.

2, the preparation of seedling

Method according to step 2 in the scheme I is carried out, and difference is: being used to inject the seedling of infecting is 5 days seedling of seed germination.

3, injection is infected and is cultivated altogether

Get the suspension of the reorganization Agrobacterium of step 1 preparation, by internal diameter is that 0.06mm, external diameter are the cotyledonary node meristematic tissue place that the tubulose syringe needle of 0.20mm is expelled to the described young shoot of the removal terminal bud of step 2 and lateral bud, each described seedling is injected 100 μ L, and it is that water was cultivated (promptly cultivating altogether) 3 days under 70% the condition that described seedling is placed 25 ℃, dark, humidity.

4, become seedling to cultivate and screening

Method according to step 4 in the scheme I is carried out.

5, PCR identifies

Method according to step 5 in the scheme I is carried out.

6, result

There is not significant difference with the result of scheme I.

The scheme III

1, the preparation of reorganization agrobacterium suspension

Method according to step 1 in the scheme I is carried out, and difference is: the final concentration of the L-halfcystine in the employed liquid nutrient medium of reorganization agrobacterium tumefaciens that suspends is 400mg/L; With respect to described liquid nutrient medium, the OD of reorganization agrobacterium suspension ₆₀₀Be 1.0.

2, the preparation of seedling

Method according to step 2 in the scheme I is carried out, and difference is: being used to inject the seedling of infecting is 10 days seedling of seed germination.

3, injection is infected and is cultivated altogether

Get the suspension of the reorganization Agrobacterium of step 1 preparation, by internal diameter is that 1mm, external diameter are the cotyledonary node meristematic tissue place that the tubulose syringe needle of 1.14mm is expelled to the described young shoot of the removal terminal bud of step 2 and lateral bud, each described seedling is injected 20 μ L, and it is that water was cultivated (promptly cultivating altogether) 3 days under 70% the condition that described seedling is placed 25 ℃, dark, humidity.

4, become seedling to cultivate and screening

Method according to step 4 in the scheme I is carried out.

5, PCR identifies

Method according to step 5 in the scheme I is carried out.

6, result

There is not significant difference with the result of scheme I.

Syringylethanone concentration is to the influence of transformation efficiency in embodiment 2, the agrobacterium suspension

1, the preparation of reorganization agrobacterium suspension

With expression vector pCambia3301(available from the Beijing DingGuo ChangSheng Biology Technology Co., Ltd, contain gus reporter gene) change agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105(over to available from sky, Beijing bounties Gene Tech. Company Limited) in, obtain containing the reorganization agrobacterium tumefaciens EHA105/pCambia3301 of gus reporter gene.

Single bacterium colony of reorganization agrobacterium tumefaciens EHA105/pCambia3301 is cultured to OD with the YEP nutrient solution (solvent is a water, and solute and concentration thereof are respectively: sodium-chlor 5g/L, yeast extract 5g/L and Tryptones 10g/L) that contains spectinomycin 50mg/L and Rifampin 25mg/L in 28 ℃ ₆₀₀Be 0.8-1.2; 4000rpm, centrifugal 10min, resuspended with following liquid nutrient medium to OD ₆₀₀Be 0.6 suspension that obtains recombinating Agrobacterium:

Solvent is a water, and solute and the final concentration in described liquid nutrient medium thereof are respectively: 1/10 B5 minimum medium inorganic salt composition, B5 minimum medium organic composition, sucrose 30g/L, 2-(N-morpholine) ethyl sulfonic acid 3.9g/L, 6-benzyladenine 1.67mg/L, L-halfcystine 1000mg/L, dithiothreitol (DTT) 154mg/L, Sulfothiorine 158mg/L, Syringylethanone 0,50,100,150,200 or 300 μ mol/L, Plant hormones regulators,gibberellins 0.1mg/L;

Described 1/10 B5 minimum medium inorganic salt composition 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, hydrochloric acid sulphur ammonium 10mg/L.

2, the preparation of seedling

Method according to step 2 in the embodiment 1 scheme I is carried out.

3, injection is infected and is cultivated altogether

Method according to step 3 in the embodiment 1 scheme I is carried out.

4, GUS dyeing detects

To be total to the seedling of cultivating through step 3, get the cotyledonary node position and carry out GUS dyeing, statistics transformation efficiency (%), be that the cotyledonary node position becomes the per-cent that blue seedling number accounts for the plant sum that infects through step 1 same concentrations Syringylethanone (AS) reorganization agrobacterium suspension, the result is as shown in table 1 and shown in Figure 4.

The painted method of above-mentioned GUS is as follows:

Cotyledonary node with 90% acetone treatment 15min, is added the GUS dye liquor and handles 16h for 37 ℃ after 0.1M phosphate buffered saline buffer (PBS) washing, after ethanol decolorization was handled, entity microscope observing was contrast with not genetically modified wild-type plant.The GUS dye liquor is formed: 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 efficient detected result

AS concentration (μ mol/L)	0	50	100	150	200	300
							Transformation efficiency (%)	10.6b	25.2b	50.0c	63.4a	72.0a	33.6b

Annotate: data are 3 multiple mean values in the table; Be marked with different lowercase person differences behind the same column result and reach 5% conspicuous level.

Embodiment 3, different soybean varieties are to the influence of transformation efficiency

Select for use respectively east farming 50, middle yellow 35, Shanxi beans 21, middle beans 33, middle beans 40, day grand No. one, cultivate rich 16 and 48 8 soybean varieties in Heihe carry out the transformation efficiency analysis.Concrete grammar is as follows:

1, the preparation of reorganization agrobacterium suspension

With expression vector pCambia3301(available from the Beijing DingGuo ChangSheng Biology Technology Co., Ltd, contain herbicide resistance gene Bar) change agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105(over to available from sky, Beijing bounties Gene Tech. Company Limited) in, obtain containing the reorganization agrobacterium tumefaciens EHA105/pCambia3301 of gus reporter gene.

Single bacterium colony of reorganization agrobacterium tumefaciens EHA105/pCambia3301 is cultured to OD with the YEP nutrient solution (solvent is a water, and solute and concentration thereof are respectively: sodium-chlor 5g/L, yeast extract 5g/L and Tryptones 10g/L) that contains spectinomycin 50mg/L and Rifampin 25mg/L in 28 ℃ ₆₀₀Be 0.8-1.2; 4000rpm, centrifugal 10min, resuspended with following liquid nutrient medium to OD ₆₀₀Be 0.6 suspension that obtains recombinating Agrobacterium:

Solvent is a water, and solute and the final concentration in described liquid nutrient medium thereof are respectively: 1/10 B5 minimum medium inorganic salt composition, B5 minimum medium organic composition, sucrose 30g/L, 2-(N-morpholine) ethyl sulfonic acid 3.9g/L, 6-benzyladenine 1.67mg/L, L-halfcystine 1000mg/L, dithiothreitol (DTT) 154mg/L, Sulfothiorine 158mg/L, Syringylethanone 200 μ mol/L, Plant hormones regulators,gibberellins 0.1mg/L;

Described 1/10 B5 minimum medium inorganic salt composition 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, hydrochloric acid sulphur ammonium 10mg/L.

2, the preparation of seedling

The soybean varieties east farming 50 of the full and surperficial no scab of picking, middle yellow 35, Shanxi beans 21, middle beans 33, middle beans 40, day grand No. one, cultivate rich 16 or Heihe 48 seeds, keep at a certain distance away and place between wetting double-deck filter paper, parcel is paper roll, be put in water planting in the plastics casing, 25 ℃, photoperiod are that illumination every day 16 hours, dark 8 hours, intensity of illumination are 100 μ molm ^-2S ^-1Condition under be cultured to seed germination 7 days, remove terminal bud and lateral bud, carry out the injection of step 3 and infect.

3, injection is infected and is cultivated altogether

Method according to step 3 in the embodiment 1 scheme I is carried out.

4, become seedling to cultivate and screening

Method according to step 4 in the scheme I is carried out.

5, PCR identifies

The step 4 of learning from else's experience is through the plant of the no considerable change of glufosinates screening, extract genomic dna, with this DNA is the pcr amplification that template is carried out resistant gene Bar, amplified production is carried out 1% agarose gel electrophoresis, the positive transfer-gen plant of plant that contains the Bar gene fragment of 440bp in the amplified production does not contain the negative transfer-gen plant of this segmental 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

Annotate: transformation efficiency=(PCR identifies positive plant number/injection seedling number) * 100%

Claims (10)

1. cultivate the method for genetically engineered soybean, change target DNA over to soya cells by agriculture bacillus mediated, it is characterized in that: described method comprises the steps:

1) after the suspension of the reorganization Agrobacterium that contains target DNA is injected at the cotyledonary node meristematic tissue place of soybean seeds sprouting seedling, described seedling is carried out common cultivation;

2) will cultivate through the directly long seedling of the described seedling that step 1) is cultivated altogether, promptly obtain having the genetically engineered soybean plant of root, stem and leaf.

2. method according to claim 1 is characterized in that: the described seedling during described the injection is 5-10 days a seedling of seed germination.

3. method according to claim 2 is characterized in that: before the described injection, comprise the step that terminal bud and the lateral bud of described seedling are removed.

4. according to arbitrary described method in the claim 1-3, it is characterized in that: the suspension of described reorganization Agrobacterium prepares according to the method that comprises the steps: described reorganization Agrobacterium is suspended in the suspension that forms described reorganization Agrobacterium in the liquid nutrient medium with following composition: solvent is a water, and solute and the final concentration in described liquid nutrient medium thereof are respectively: 1/10 B5 minimum medium inorganic salt composition, B5 minimum medium organic composition, sucrose 30g/L, 2-(N-morpholine) ethyl sulfonic acid 3.9g/L, 6-benzyladenine 1.67mg/L, L-halfcystine 400-1000mg/L, dithiothreitol (DTT) 154mg/L, Sulfothiorine 158mg/L, Syringylethanone 0-300 μ mol/L, Plant hormones regulators,gibberellins 0.1mg/L;

Described 1/10 B5 minimum medium inorganic salt composition 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, hydrochloric acid sulphur ammonium 10mg/L.

5. method according to claim 4 is characterized in that: compare the OD of the suspension of described reorganization Agrobacterium with described liquid nutrient medium ₆₀₀Value is 0.4-1.0; The injection volume of the suspension of described reorganization Agrobacterium is each described seedling 20-100 μ L.

6. according to arbitrary described method in the claim 1-5, it is characterized in that: described injection is that 0.06mm-1mm, external diameter are the syringe needle realization of 0.20mm-1.14mm by internal diameter.

7. according to arbitrary described method in the claim 1-6, it is characterized in that: described Agrobacterium is an agrobacterium tumefaciens, specifically can be agrobacterium tumefaciens EHA105.

8. according to arbitrary described method in the claim 1-7, it is characterized in that: described culture condition was altogether cultivated 3 days for water under 25 ℃ of temperature, dark condition.

9. an Agrobacterium is infected the liquid nutrient medium that plant is used, it is characterized in that: the solvent of described liquid nutrient medium is a water, and solute and the final concentration in described liquid nutrient medium thereof are respectively: 1/10 B5 minimum medium inorganic salt composition, B5 minimum medium organic composition, sucrose 30g/L, 2-(N-morpholine) ethyl sulfonic acid 3.9g/L, 6-benzyladenine 1.67mg/L, L-halfcystine 400-1000mg/L, dithiothreitol (DTT) 154mg/L, Sulfothiorine 158mg/L, Syringylethanone 0-300 μ mol/L, Plant hormones regulators,gibberellins 0.1mg/L;

Described 1/10 B5 minimum medium inorganic salt composition 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, hydrochloric acid sulphur ammonium 10mg/L.

10. substratum according to claim 9 is characterized in that: described plant is monocotyledons or dicotyledons, and described dicotyledons specifically can be soybean.

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