CN102002512A - Genetic transformation method for soybean - Google Patents

Abstract

The invention discloses a genetic transformation method for soybean. The method provided by the invention comprises the following steps of: delivering an exogenous gene into hypocotyl of the soybean to obtain transgenic soybean, wherein the hypocotyl of the soybean contains apical meristem of 0.5 to 1cm; and preparing the hypocotyl of the soybean according to the following steps of: germinating soybean seeds for 5 to 6 days, removing seed coats, cotyledons and terminal buds to obtain the hypocotyl of the soybean containing apical meristem of 0.5 to 1cm. The invention provides the method for establishing a genetic transformation system of the soybean by using the hypocotyl as an receptor, so that adventitious buds can be quickly and efficiently produced in a shorter time, and the method is used for a basic operating system of genetic engineering of the soybean and has important theory and practical significance for improvement on quality of the soybean.

Description

A kind of soybean heredity method for transformation

Technical field

The present invention relates to plant genetic engineering field, relate in particular to a kind of soybean heredity method for transformation.

Background technology

Soybean [Glycine max (L.) Merr.] belongs to annual leguminous herbaceous plant, and China is the country of origin of soybean, the history of existing more than 4700 year manufacturing soybean.Soybean is important oil crops and food crop, is the important source of edible oil and vegetable-protein in the world.Yet the difficulty of soybean regeneration and genetic transformation has hindered the further raising of soybean economical character and the progress of functional genome research.Up to now, what obtain from the soybean expression library surpasses 300,000 expressed sequence tag (expressed sequence tages, ESTs) gene clone of products for further and functional verification, these est sequences are being represented with the soybean organ and are being built up, a big genoid of developmental condition, genotype and envrionment conditions etc.The soybean heredity resource is demanded people urgently and is gone exploitation, and is used for improveing the cultivated soybean.

With respect to crops such as corn, paddy rice, the conversion of soybean progress is slower.Hinchee (1988) reports the acquisition of soybean transgene plant the earliest, and the soybean transformation frequency of having reported so far is still lower.Transgenic method commonly used in soybean transgene is particle bombardment and agrobacterium-mediated transformation, and it is less that other method for transformation is used.McCabe etc. (1988) reported first utilize micropellet bombardment method success soybean transformation terminal bud meristematic tissue to obtain transfer-gen plant, but all be mosaic.After this report adopts this system to carry out the later stage screening by selecting to contain by the plant of transformant, has obtained non-chimeric transfer-gen plant.Because the micropellet bombardment method is subjected to the restriction of target tissue unlike agrobacterium co-cultivation, its report that is applied to other target cell tissues also occurs subsequently.Finer and McMullen (1991) reported first is with particle bombardment transformant somatic embryo sexual cell group and obtained transfer-gen plant, and this system is able to fast development later on, becomes the means that adopt in most of laboratories.The particle bombardment operation is subjected to the influence of acceptor material less, and transformation efficiency is higher relatively, and easy and simple to handle, the degree of controllability height.But this method imports the dna molecular amount generally is not higher than 10kb, and integrates in a plurality of sites of Plant Genome easily and cause copy number higher, easily causes gene silencing.Since (1988) such as Hinchee utilized the success of Agrobacterium-mediated Transformation soybean cotyledon node, what soybean transformed generally employing all was this based on agriculture bacillus mediated cotyledonary node meristematic tissue regeneration system rapidly.Because T-DNA is integrated into merismatic frequency, to the efficiency of selection of transformant, adventitious shoot regeneration frequency and plant regeneration frequency etc. are not high, agriculture bacillus mediated soybean cotyledon node transformation frequency is very low always.People have carried out many optimizations to the soybean conversion process in the test.In the process that soybean heredity transforms, select those to cause the agrobacterium strains of strong toxicity as far as possible.Ultrasonication (SAATsonication-assisted Agrobacterium-mediated transformation) and auxiliary micropellet bombardment also can increase the number of contaminating the site, adopting embryonal suspension system, unmature subleaf or cotyledonary node etc. is target tissue, make Agrobacterium pass through cells of superficial layer and enter deep layer and be more prone to, can significantly improve transformation efficiency.Though agriculture bacillus mediated soybean cotyledon node transformation frequency is also very low, the test operation circulation ratio is not high yet, and this method is still adopted by most of laboratories and company and is used for carrying out transgenic research.With respect to the somatic embryo approach, agriculture bacillus mediated cotyledonary node approach is drawn materials conveniently, culture cycle short (general 8 weeks obtain regeneration plant); And required test conditions is simple, need not equipment such as particle gun, and it is cheap relatively to obtain the transfer-gen plant cost.But than higher, operator's skill level and experience often depended in the success of test to the entire operation process to operator's technical requirements.

In recent years, Liu et al. (2004) utilizes the sharp meristematic tissue of agriculture bacillus mediated embryo system, has obtained higher transformation frequency.Paz et al. (2006) reports with an agriculture bacillus mediated sub-approach of semispecies, has improved the transformation frequency of cotyledonary node approach.But one of soybean difficult plant transformed of still generally acknowledging up to the present.This is that repeatable poor, transformation frequency differs greatly between genotype because the regeneration difficulty is cultivated by soyabean tissue, makes that utilizing genetic engineering means that soybean is carried out genetic improvement is restricted.Therefore, have only the good transgene receptor system that sets up, and in conjunction with suitable transgenic method, just might the render transgenic technology in the cultivation of the character improvement of soybean, new variety, play a role, and set up a new way that is different from traditional breeding method.

Summary of the invention

The purpose of this invention is to provide a kind of soybean heredity method for transformation.

Method provided by the invention comprises the steps: 1) use the reorganization Agrobacterium solution that contains foreign gene to infect the hypocotyl of soybean, obtain infecting the back hypocotyl;

2) the back hypocotyl of infecting that step 1) is obtained carries out common cultivation and obtains common cultivations hypocotyl afterwards;

3) with step 2) obtain cultivate the back hypocotyl and carry out hypocotyl after the bud inducing culture obtains bud and induces altogether;

The hypocotyl of the band bud that 4) step 3) is obtained carries out the hypocotyl that screening and culturing obtains screening the back survival;

5) take off indefinite bud the hypocotyl that after the screening that step 4) obtains, survives, indefinite bud is carried out root culture, obtain the genetically engineered soybean seedling.

The hypocotyl of described soybean is the long hypocotyl of 0.5cm-1cm with apical meristem, and the hypocotyl of described soybean is specially 0.5cm, 0.8cm or the long hypocotyl of 1cm with apical meristem;

The hypocotyl of described acceptor soybean is prepared as follows: soybean seeds was sprouted 5 days-6 days, removed kind of a skin, cotyledon, terminal bud, obtained having the long hypocotyl of 0.5cm-1cm of apical meristem;

The hypocotyl of described acceptor soybean specifically is prepared as follows: soybean seeds was sprouted 5 days or 6 days, removed kind of a skin, cotyledon, terminal bud, obtained having 0.5cm, 0.8cm or the long hypocotyl of 1cm of apical meristem.

Described acceptor soybean seeds is sprouted after 5 days-6 days and is had following structure: have kind of a skin, cotyledon, terminal bud, hypocotyl and root, the outer skin of planting breaks, and cotyledon expands, and terminal bud is arranged in the cotyledon, the primary leaf of terminal bud does not also expose under cotyledon, the cotyledon hypocotyl, under the hypocotyl root is arranged.

In the step 1), the described reorganization Agrobacterium solution that contains foreign gene is for to be prepared as follows: the described reorganization Agrobacterium that contains foreign gene is resuspended in liquid altogether in the culture medium, transfer to the OD value and be 0.3-0.8,25 ℃ leave standstill 1h, and Agrobacterium solution obtains recombinating; Described OD value is specially 0.3,0.4,0.5,0.6 or 0.8;

Described liquid culture medium altogether is prepared as follows: add 2-morphine acid-ethyl sulfonic acid in the 1/2MS substratum, Sulfothiorine, the L-halfcystine, dithiothreitol (DTT), Syringylethanone, 6-benzyladenine, Silver Nitrate and sucrose, obtain described liquid culture medium altogether, described 2-morphine acid-ethyl sulfonic acid is 3.9g/L in the concentration that described liquid is total in the culture medium, described Sulfothiorine is 0.2g/L in the concentration that described liquid is total in the culture medium, described L-halfcystine is 400mg/L in the concentration that described liquid is total in the culture medium, described dithiothreitol (DTT) is 0.154g/L in the concentration that described liquid is total in the culture medium, described Syringylethanone is 200 μ mol/L in the concentration that described liquid is total in the culture medium, described 6-benzyladenine is 4mg/L in the concentration that described liquid is total in the culture medium, described Silver Nitrate is 5mg/L in the concentration that described liquid is total in the culture medium, described sucrose is 30 in the concentration that described liquid is total in the culture medium, 000mg/L, described the liquid pH of culture medium altogether are 5.6;

Step 2) in, describedly cultivate used substratum altogether and be prepared as follows: add agar in the culture medium altogether to described liquid and obtain the used substratum of common cultivation, described agar is 6 in described concentration of cultivating altogether in the used substratum, 000mg/L, the described pH that cultivates used substratum altogether is 5.6;

In the step 3), the used substratum of described bud inducing culture is prepared as follows: add 6-benzyladenine in the 1/2MS substratum, indolebutyric acid, sucrose, Pyocianil, Reflin, Silver Nitrate and agar, obtain the used substratum of described bud inducing culture, the concentration of described 6-benzyladenine in the used substratum of described bud inducing culture is 4mg/L, the concentration of described indolebutyric acid in the used substratum of described bud inducing culture is 0.2mg/L, the concentration of described sucrose in the used substratum of described bud inducing culture is 30,000mg/L, the concentration of described Pyocianil in the used substratum of described bud inducing culture is 300mg/L, the concentration of described Reflin in the used substratum of described bud inducing culture is 300mg/L, the concentration of described Silver Nitrate in the used substratum of described bud inducing culture is 5mg/L, the concentration of described agar in the used substratum of described bud inducing culture is 8,000mg/L, the pH of the used substratum of described bud inducing culture is 5.8;

In the step 4), the used substratum of described screening and culturing is prepared as follows: add kantlex in the used substratum of described bud inducing culture, obtain the used substratum of described screening and culturing, the concentration of described kantlex in the used substratum of described screening and culturing is 100mg/L, and the pH of the used substratum of described screening and culturing is 5.8;

In the step 5), the used substratum of described root culture is prepared as follows: add sucrose in the 1/2MS substratum, Reflin, kantlex and agar, obtain the used substratum of described root culture, the concentration of described sucrose in the used substratum of described root culture is 20,000mg/L, the concentration of described Reflin in the used substratum of described root culture is 150mg/L, the concentration of described kantlex in the used substratum of described root culture is 10mg/L, the concentration of described agar in the used substratum of described root culture is 8,000mg/L, the pH value of the used substratum of described root culture is 5.6.

In the step 1), the described time of infecting is 0.5h-8h; The described mode that infects is: the hypocotyl of described acceptor soybean is immersed the reorganization Agrobacterium solution shaking culture that contains foreign gene; The described time of infecting is specially 0.5h, 1h, 1.5h, 2h, 3h, 4h, 5h, 6h, 7h or 8h;

Step 2) in, described temperature of cultivating altogether is 22 ℃-25 ℃, and described temperature of cultivating altogether is specially 22 ℃, 23 ℃ or 25 ℃, and described the cultivation altogether to dark cultivated, and the described time of cultivating altogether is 2 days-5 days; The described time of cultivating altogether was specially 2 days, 3 days, 4 days or 5 days;

In the step 3), 25 ℃-28 ℃ of the temperature of described bud inducing culture, the temperature of described bud inducing culture is specially 25 ℃, 27 ℃ or 28 ℃, and intensity of illumination is 70 μ Em ^-2S ^-1, photoperiod control 18/6h (light/dark), incubation time is 6 days;

In the step 4), 25 ℃-28 ℃ of the temperature of described screening and culturing, the temperature of described screening and culturing is specially 25 ℃, 27 ℃ or 28 ℃, and intensity of illumination is 70 μ Em ^-2S ^-1, photoperiod control 18/6h light/dark, and subculture is once weekly; The time of described screening and culturing was 4 weeks;

In the step 5), 25 ℃-28 ℃ of the temperature of described root culture, the temperature of described root culture is specially 25 ℃, 27 ℃ or 28 ℃, and intensity of illumination is 70 μ Em ^-2S ^-1, photoperiod control 18/6h light/dark, incubation time 15 days.

In the step 5), the length of described indefinite bud is 2.5cm-4cm, and the length of described indefinite bud is specially 2.5cm, 3cm or 4cm.

In step 2) after, before the step 3), also comprise successively with distilled water and contain Pyocianil and the Reflin liquid described hypocotylar step in back of cultivating altogether of culture medium flushing altogether;

In the described step 5), the described indefinite bud that takes off downcuts indefinite bud for the base portion from indefinite bud; Described take off the step of indefinite bud after, described indefinite bud is carried out the root culture step before, comprise that also it is the step of soaking in the indolebutyric acid aqueous solution of 2mg/L that the shearing end that will downcut indefinite bud immerses concentration;

Described successively with distilled water with contain the described hypocotylar step in back of cultivating altogether of Pyocianil and Reflin liquid nutrient medium flushing, operation as follows: earlier with sterile distilled water flushing 3 times, then with containing Pyocianil and Reflin liquid nutrient medium flushing 4 times, the described liquid that contains Pyocianil and Reflin is total to culture medium and is prepared as follows: add Pyocianil and Reflin in the culture medium altogether to described liquid, the liquid that contains Pyocianil and Reflin that obtains is culture medium altogether, described Pyocianil is 300mg/L in the concentration that the described liquid that contains Pyocianil and Reflin is total in the culture medium, and described Reflin is 300mg/L in the concentration that the described liquid that contains Pyocianil and Reflin is total in the culture medium;

It is in the step of soaking in the indolebutyric acid aqueous solution of 2mg/L that the described shearing end that will downcut indefinite bud immerses concentration, and described soak time is 2min-5min; Described soak time is specially 2min, 3min, 4min or 5min; The position of the described indefinite bud of described cutting-out is a base portion of being close to indefinite bud;

In the described method, after described step 5), also comprise with having of obtaining of root culture greater than the seedling replanting of the adventive root of 2cm in composite soil, cultivated for 1 week, obtain the step of genetically engineered soybean plant, described composite soil is made up of turfy soil and vermiculite, and the mass ratio of described turfy soil and vermiculite is 1: 1, the length of described adventive root is preferably 2cm-4cm, is specially 2cm, 3cm or 4cm.

Among the hypocotyl preparation method of described soybean, the used substratum of described sprouting is prepared as follows: add sucrose and agar in the 1/2MS substratum, obtain described sprouting and cultivate used substratum, the concentration that described sucrose is cultivated in the used substratum in described sprouting is 15,000mg/L, the concentration that described agar is cultivated in the used substratum in described sprouting is 7500mg/L, and the pH of the substratum that described sprouting is used is 5.8;

The temperature that described sprouting is cultivated is 22 ℃-25 ℃, and the temperature that described sprouting is cultivated is specially 22 ℃, 23 ℃ or 25 ℃, and intensity of illumination is 120 μ Em ^-2S ^-1, periodicity of illumination is 18h/6h illumination/dark.

The described reorganization Agrobacterium that contains foreign gene is following 1) or 2):

1), carrier pCAMBIA2301 is imported the reorganization Agrobacterium that agrobacterium tumefaciens lba4404 obtains

2), carrier pCAMBIA2301 is imported the reorganization Agrobacterium that agrobacterium tumefaciens EHA105 obtains.

The 1/2MS substratum is prepared as follows: add ammonium nitrate, saltpetre, calcium chloride, sal epsom, potassium primary phosphate, boric acid, manganous sulfate, zinc sulfate, potassiumiodide, Sodium orthomolybdate, copper sulfate, cobalt chloride, glycine, VITMAIN B1, vitamin B6, nicotinic acid, inositol, ferrous sulfate and b diammonium disodium edta in deionized water.The concentration of described ammonium nitrate in described 1/2MS substratum is 825mg/L, the concentration of saltpetre in described 1/2MS substratum is 950mg/L, the concentration of described calcium chloride in described 1/2MS substratum is 220mg/L, the concentration of described sal epsom in described 1/2MS substratum is 185mg/L, the concentration of described potassium primary phosphate in described 1/2MS substratum is 95mg/L, the concentration of described boric acid in described 1/2MS substratum is 3.1mg/L, the concentration of described manganous sulfate in described 1/2MS substratum is 11.15mg/L, the concentration of described zinc sulfate in described 1/2MS substratum is 4.3mg/L, the concentration of described potassiumiodide 0.415mg/L in described 1/2MS substratum is, the concentration of described Sodium orthomolybdate 0.125mg/L in described 1/2MS substratum is, the concentration of described copper sulfate in described 1/2MS substratum is 0.0125mg/L, the concentration of described cobalt chloride in described 1/2MS substratum is 0.0125mg/L, the concentration of described glycine in described 1/2MS substratum is 1mg/L, the concentration of described VITMAIN B1 in described 1/2MS substratum is 0.05mg/L, the concentration of described vitamin B6 in described 1/2MS substratum is 0.25mg/L, the concentration of described nicotinic acid in described 1/2MS substratum is 0.25mg/L, the concentration of described inositol in described 1/2MS substratum is 50mg/L, the concentration of described ferrous sulfate in described 1/2MS substratum is 13.975mg/L, and the concentration of described b diammonium disodium edta in described 1/2MS substratum is 18.625mg/L.

Of the present invention experiment showed, added the incidence that high density 6-benzyladenine (BAP) can be induced explant meristematic tissue propagation generation indefinite bud in substratum; In substratum, add the generation that silver ions can promote the single explant polygerm of soybean significantly, make the evoking adventive bud overall number significantly increase; Reach and sprout manyly, the regrowth ability is strong, compares with common agriculture bacillus mediated method for transformation, and transformation efficiency increases substantially.The hypocotyl that utilizes that the invention provides is set up the method for soybean heredity transformation system for acceptor, but rapidly and efficiently produce indefinite bud within a short period of time, the basic operating system that is used for the soybean gene engineering, improvement has important theory and realistic meaning for soybean quality.

Description of drawings

Fig. 1 be in the bud inducing culture different 6-benzyladenines (BAP) concentration to the diagrammatic sketch of the influence of soybean hypocotyl regeneration frequency

Fig. 2 is the diagrammatic sketch in the comparison of adventitious bud inducing stage soybean hypocotyl and cotyledonary node regeneration frequency

Fig. 3 adds 5mg/L Silver Nitrate (AgNO in the bud inducing culture ₃) to the diagrammatic sketch of the influence of soybean hypocotyl explant adventitious shoot regeneration

Fig. 4 is the diagrammatic sketch of the influence that Silver Nitrate transforms soybean in different agrobacterium strains and the substratum

Fig. 5 is the diagrammatic sketch of bacterial concentration to the influence of soybean hypocotyl transient expression frequency

Fig. 6 is the diagrammatic sketch of immerged time to the influence of agriculture bacillus mediated soybean hypocotyl T-DNA transformation efficiency

Fig. 7 is that different incubation times altogether are to the diagrammatic sketch of soybean hypocotyl gus gene transient expression frequency influence

Fig. 8 is the diagrammatic sketch of antioxidant to the influence of brownization of hypocotyl in the tissue culture procedures and gus gene expression

Fig. 9 is the diagrammatic sketch of Syringylethanone (AS) concentration in the culture medium altogether to the influence of gus gene transient expression frequency

Figure 10 is the part physical map of plasmid pCAMBIA2301

Figure 11 is the photo that gus gene is expressed at agriculture bacillus mediated soybean hypocotyl

Figure 12 is that agriculture bacillus mediated hypocotyl transforms the dyeing photo that obtains the GUS positive plant

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.

It is all glad through company of section available from Beijing to test related medicine among the following embodiment, and worker company is given birth in Sigma company and Shanghai.

The influence factor that embodiment 1, soybean hypocotyl regeneration plant obtain

One, BAP concentration is to the influence of hypocotyl regenerated adventitious bud frequency

1, the acquisition of acceptor soybean hypocotyl

Test materials: the black farming 44 of soybean (Glycine max), be documented in full be group, Du Weiguang, Chen Yi, Luan Xiaoyan, Liu Xinlei, Wang Fengli, the seed selection of the black farming 44 of new soybean varieties and Different Ways of Planting are to the influence of its yield and quality, the Heilungkiang agricultural sciences, 2004,5:1-3, in, the public can obtain from Institute of Botany, Chinese Academy of Sciences.

Soybean seeds surface sterilization: select anosis, full soybean seeds and be tiled in the uncovered culture dish, put into built-in one moisture eliminator that fills 100mL clorox stoste beaker, slowly add the 4mL concentrated hydrochloric acid along beaker then, the close drying device places the stink cupboard 18h that sterilizes.Seed after the sterilization is placed in the germination medium (500mg/L agar, pH 5.8 for 1/2MS minimum medium+15,000mg/L sucrose+7), places 25 ℃, 120 μ Em ^-2S ^-1Cultivate 5d under the condition of light intensity (periodicity of illumination 18/6h).

Soybean hypocotyl: when cultivating 5d, sprout soybean peel this moment and break, primary leaf (primary leaves) does not also expose cotyledon.In Bechtop, take out soybean, carefully remove kind of a skin, cotyledon is peeled off.With scalpel terminal bud is excised then, the hypocotyl that cuts the 0.5cm with apical meristem is as the acceptor soybean hypocotyl.

2, bud inducing culture

The soybean hypocotyl that cuts is transferred to bud inducing culture (the 1/2MS minimum medium that contains 1mg/L, 2mg/L, 3mg/L, 4mg/L or 5mg/L 6-benzyladenine respectively, 0.2mg/L indolebutyric acid, 30,000mg/L sucrose, the 300mg/L Pyocianil, the 300mg/L Reflin, 8,000mg/L agar, 1mg/L, 2mg/L, 3mg/L, 4mg/L or 5mg/L 6-benzyladenine) on carry out the bud inducing culture, the pH of substratum is 5.8, and culture temperature is 27 ℃, and intensity of illumination is 70 μ Em ^-2S ^-1, photoperiod control 18/6h cultivates 6d, obtains indefinite bud respectively, and the regeneration frequency of statistics indefinite bud, regeneration frequency are regenerate explant number/explant number * 100% of indefinite bud.The number of explant is 50, experiment triplicate, results averaged.

The result is:

Contain on the bud inducing culture of 1mg/L 6-benzyladenine, the regeneration frequency of indefinite bud is 28.2%; The regeneration frequency that wherein is less than 3 indefinite buds is 13.0%, is 15.2% more than the regeneration frequency of 4 indefinite buds;

Contain on the bud inducing culture of 2mg/L 6-benzyladenine, the regeneration frequency of indefinite bud is 41%; The regeneration frequency that wherein is less than 3 indefinite buds is 23.5%, is 17.5% more than the regeneration frequency of 4 indefinite buds;

Contain on the bud inducing culture of 3mg/L 6-benzyladenine, the regeneration frequency of indefinite bud is 86.7%; The regeneration frequency that wherein is less than 3 indefinite buds is 41.2%, is 45.3% more than the regeneration frequency of 4 indefinite buds;

Contain on the bud inducing culture of 4mg/L 6-benzyladenine, the regeneration frequency of indefinite bud is 85.3%; The regeneration frequency that wherein is less than 3 indefinite buds is 41.6%, is 43.7% more than the regeneration frequency of 4 indefinite buds;

Contain on the bud inducing culture of 5mg/L 6-benzyladenine, the regeneration frequency of indefinite bud is 82.0%; The regeneration frequency that wherein is less than 3 indefinite buds is 39.6%, is 42.4% more than the regeneration frequency of 4 indefinite buds;

Map as shown in Figure 1, as seen from the figure, the 4mg/L 6-benzyladenine significantly improves the regeneration frequency of indefinite bud.The concentration of substratum interpolation 6-benzyladenine has obvious effect to the number and the frequency of explant indefinite bud.Contain under the 4mg/L6-benzyladenine condition, soybean hypocotyl can efficiently be induced the generation of polygerm, and its regeneration frequency significantly improves.

Cultivate on the 6-benzyladenine substratum of greater concn, though the result increases for the ratio that single explant produces polygerm, total regeneration frequency descends on the contrary to some extent.

Two, the hypocotyl regeneration system is with the comparison of cotyledonary node regeneration system

1, the acquisition of acceptor soybean hypocotyl

Test materials: identical with above-mentioned one;

Soybean seeds surface sterilization: identical with above-mentioned one;

Soybean cotyledon node explant: take out the soybean seeds of on germination medium, cultivating 5 days, peel off kind of a skin, cut root and most hypocotyl, only stay hypocotyl part apart from cotyledonary node 3mm.Along hilum cotyledon is separated, with primary leaf and auxilliary bud complete resection, do injury reason (drawing 10 times perpendicular to cotyledon) at the cotyledonary node place with scalpel then, make the strong meristematic tissue of meristematic capacity expose, the cotyledonary node that obtains is as the soybean cotyledon node explant.

2, bud inducing culture: basic identical with above-mentioned one method, different is that wherein bud inducing culture based formulas is as follows: the 1/2MS minimum medium, 0.2mg/L indolebutyric acid, 30,000mg/L sucrose, the 300mg/L Pyocianil, the 300mg/L Reflin, 8,000mg/L agar, the 4mg/L 6-benzyladenine, the soybean plant strain (cotyledonary node) that obtains regenerating group.

(fate herein is the time of bud inducing culture) contained regeneration soybean plant strain (hypocotyl) group and the above-mentioned regeneration soybean plant strain (cotyledonary node) that obtains that the bud inducing culture of 4mg/L 6-benzyladenine obtains by embodiment 1 and organized the indefinite bud number that obtains at the bud inducing culture to add up 5,10,15,20,25 days respectively; The regeneration frequency of statistics indefinite bud, regeneration frequency are regenerate explant number/explant number * 100% of indefinite bud.The number of explant is 50, experiment triplicate, results averaged.

The result is: in the time of 5 days, the regeneration frequency of indefinite bud is respectively 0 and 6.0% at the bud inducing culture for regeneration soybean plant strain (cotyledonary node) group and regeneration soybean plant strain (hypocotyl) group; The regeneration frequency that wherein is less than 3 indefinite buds is respectively 0 and 5.8%, is respectively 0 and 0.2% more than the regeneration frequency of 4 indefinite buds;

In the time of 10 days, the regeneration frequency of indefinite bud is respectively 4.1% and 51.3% at the bud inducing culture for regeneration soybean plant strain (cotyledonary node) group and regeneration soybean plant strain (hypocotyl) group; The regeneration frequency that wherein is less than 3 indefinite buds is respectively 4.1% and 31.6%, is respectively 0 and 19.7% more than the regeneration frequency of 4 indefinite buds;

In the time of 15 days, the regeneration frequency of indefinite bud is respectively 8.5% and 63.5% at the bud inducing culture for regeneration soybean plant strain (cotyledonary node) group and regeneration soybean plant strain (hypocotyl) group; The regeneration frequency that wherein is less than 3 indefinite buds is respectively 7.5% and 35.7%, is respectively 1% and 27.8% more than the regeneration frequency of 4 indefinite buds;

In the time of 20 days, the regeneration frequency of indefinite bud is respectively 21.0% and 82.5% at the bud inducing culture for regeneration soybean plant strain (cotyledonary node) group and regeneration soybean plant strain (hypocotyl) group; The regeneration frequency that wherein is less than 3 indefinite buds is respectively 15.4% and 42.2%, is respectively 4.6% and 40.3% more than the regeneration frequency of 4 indefinite buds;

In the time of 25 days, the regeneration frequency of indefinite bud is respectively 40.5% and 82.7% at the bud inducing culture for regeneration soybean plant strain (cotyledonary node) group and regeneration soybean plant strain (hypocotyl) group; The regeneration frequency that wherein is less than 3 indefinite buds is respectively 22.2% and 39.8%, is respectively 18.3% and 42.9% more than the regeneration frequency of 4 indefinite buds;

Map as shown in Figure 2, soybean hypocotyl can be induced the regeneration of high frequency adventitious bud high frequency in very short time, and the ratio of each explant induction polygerm (greater than 3 indefinite buds) also will be higher than the cotyledonary node regeneration system simultaneously.The soybean hypocotyl regeneration frequency is 2.2 times of cotyledonary node regeneration frequency.Find out that from figure for the cotyledonary node regeneration system, the soybean hypocotyl regeneration system has higher regeneration frequency, show that hypocotyl can rapidly and efficiently produce indefinite bud within a short period of time, thereby help further conversion operation.

Three, Silver Nitrate influences the soybean hypocotyl regenerated

1, the acquisition of acceptor soybean hypocotyl

Test materials: identical with above-mentioned one;

Soybean seeds surface sterilization: identical with above-mentioned one;

Acceptor soybean hypocotyl: identical with above-mentioned one;

2, bud inducing culture: basic identical with above-mentioned one method, different is to adopt bud inducing culture 1 to cultivate to obtain control group; Utilize bud inducing culture 2 to cultivate, obtain experimental group.

Bud inducing culture 1 prescription is as follows:: 1/2MS minimum medium, 0.2mg/L indolebutyric acid, 30,000mg/L sucrose, 300mg/L Pyocianil, 300mg/L Reflin, 8,000mg/L agar, 4mg/L 6-benzyladenine.

Bud inducing culture 2 prescriptions are as follows: 1/2MS minimum medium, 0.2mg/L indolebutyric acid, 30,000mg/L sucrose, 300mg/L Pyocianil, 300mg/L Reflin, 8,000mg/L agar, 4mg/L 6-benzyladenine, 5mg/L Silver Nitrate.

Add up the indefinite bud number that control group and experimental group obtain at the bud inducing culture respectively; The regeneration frequency of statistics indefinite bud, regeneration frequency are regenerate explant number/explant number * 100% of indefinite bud.The number of explant is 50, experiment triplicate, results averaged.

The result is as follows:

Total indefinite bud number of experimental group is 360, and regeneration frequency is 83.0%, and the regeneration frequency that wherein is less than 3 indefinite buds is 21.2%, is 61.8% more than the regeneration frequency of 4 indefinite buds;

Total indefinite bud number of control group is 240, and regeneration frequency is 81.2%, and the regeneration frequency that wherein is less than 3 indefinite buds is 42.3%, is 38.9% more than the regeneration frequency of 4 indefinite buds;

With the The above results mapping as shown in Figure 3, bar shaped post numbering is respectively 1 and 2 among the figure, and wherein 1 expression does not add Silver Nitrate processing (control group), and 2 expressions add Silver Nitrate and handle (experimental group).As can be seen, by add the generation that the 5mg/L Silver Nitrate has promoted single explant polygerm greatly in the bud inducing culture, total indefinite bud number significantly increases.But, it is pointed out that add Silver Nitrate after hypocotyl regeneration frequency (obtaining the explant number of regeneration bud) do not have noticeable change.

Four, the influence factor of taking root of regeneration bud

1, the acquisition of acceptor soybean hypocotyl

Test materials: identical with above-mentioned one;

Soybean seeds surface sterilization: identical with above-mentioned one;

Acceptor soybean hypocotyl: identical with above-mentioned one;

2, bud inducing culture: basic identical with above-mentioned one method, different is to adopt bud inducing culture 2 to cultivate:

Bud inducing culture 2 prescriptions are as follows: 1/2MS minimum medium, 0.2mg/L indolebutyric acid, 3% sucrose, 300mg/L Pyocianil, 300mg/L Reflin, 8,000mg/L agar, 4mg/L 6-benzyladenine, 5mg/L Silver Nitrate.

3, regeneration plant takes root and transplanting

When treating indefinite bud length greater than 2.5cm (hypocotyl was cultivated for 4 weeks on screening and culturing), the base portion of being close to indefinite bud downcuts it, the indefinite bud that downcuts is divided into two groups carries out following processing:

Experiment secondary root group: the otch of the indefinite bud that downcuts is immersed 2min in the 2mg/L indolebutyric acid aqueous solution, carry out root culture then, what obtain is the experiment secondary root.

Contrast secondary root group 1: the indefinite bud that downcuts is directly inserted carry out root culture in the root media, what obtain is contrast secondary root 1.

Contrast secondary root group 2: the indefinite bud that downcuts is directly inserted carry out root culture in the root media 2, root culture substratum 2 is for to add indolebutyric acid in the root media to, making the concentration of indolebutyric acid in root media is 2mg/L, and what obtain is contrast secondary root 2.

The prescription of root media is as follows: the 1/2MS minimum medium, and 2% sucrose, the 150mg/L Reflin, the 10mg/L kantlex, 8,000mg/L agar is formed, and the pH value is 5.6.

The number of explant is 20, experiment triplicate, results averaged.

The result is:

Experiment secondary root group: root culture is after 15 days, and 1 indefinite bud grows the secondary root of 3-7 bar, and rooting rate reaches 97%.

Contrast secondary root group 1: root culture is after 15 days, and 1 indefinite bud grows the secondary root of 1-2 bar, and rooting rate reaches 95%.

Contrast secondary root group 2: root culture is after 15 days, and 1 indefinite bud grows 0 secondary root, and rooting rate is 0.

From as can be seen above-mentioned, cultivate at the elongation bud that does not contain on the hormone culture-medium, the bar number of inducing adventitious root will lack, and adventive root to go out to educate the time also slow.If indolebutyric acid is directly added root media, then often cause otch callus hyper-proliferative, influenced inducing of adventive root on the contrary.

Five, the influence factor of soybean hypocotyl conversion

A: Agrobacterium kind and Silver Nitrate influence

1, the acquisition of acceptor soybean hypocotyl

Test materials: identical with above-mentioned one;

Soybean seeds surface sterilization: identical with above-mentioned one;

Acceptor soybean hypocotyl: identical with above-mentioned one;

2, Agrobacterium preparation and conversion:

1) Agrobacterium preparation

The competent preparation of Agrobacterium: picking soil Agrobacterium EHA105 (Hood EE, Gelvin SB, Melchers LS, Hoekema A (1993) New Agrobacterium helper plasmids for gene transfer toplants.Transgenic Res 2:208-218, the public can obtain from Institute of Botany, Chinese Academy of Sciences.) single colony inoculation contains the 50mg/L Rifampin in 5mL, in the YEP liquid nutrient medium of 50mg/L Streptomycin sulphate.Described YEP liquid nutrient medium is to prepare as follows: add Tryptones, yeast extract, sodium-chlor in distilled water; The concentration of described Tryptones in described YEP liquid nutrient medium is 10,000mg/L, the concentration of described yeast extract in described YEP liquid nutrient medium is 10,000mg/L, the concentration of described sodium-chlor in described YEP liquid nutrient medium is 5,000mg/L, the pH value of described YEP liquid nutrient medium is 7.0.

28 ℃, 250rpm shaking culture spend the night; Be inoculated in 40mL by 1: 100 and contain the 50mg/L Rifampin, continue to cultivate 4-6h in the YEP liquid nutrient medium of 50mg/L Streptomycin sulphate; 4 ℃, 5, the centrifugal 10min of 000rpm removes supernatant; CaCL with 600 μ L ice precooling 0.05mol/L ₂The solution washing thalline; 4 ℃, 5, the centrifugal 10min of 000rpm removes supernatant; CaCL with 200 μ L ice precooling 0.05mol/L ₂Resuspended thalline obtains Agrobacterium EHA105 competence, in 4 ℃ of preservations.

Plasmid is to the conversion of Agrobacterium: pCAMBIA2301 (www.cambia.org.au) plasmid DNA (this plasmid contains selectable marker gene NPTII and gus reporter gene) that adds about 0.5 μ g in Agrobacterium EHA105 competence, mixing is placed 5min on ice gently; Place liquid nitrogen 3min then; Be transferred to 37 ℃ of incubation 5min rapidly; Add 500 μ L YEP liquid nutrient mediums, 28 ℃ of shaking culture 5-6h; 5, the centrifugal 3min of 000rpm removes most of supernatant, surplus 200 μ L, suspension thalline; Evenly coat the YEP that contains 50mg/L Rifampin, 50mg/L Streptomycin sulphate and 50mg/L kantlex and select on the flat board, be inverted for 28 ℃ and cultivated two days, obtain single bacterium colony.Picking list colony inoculation contains the 50mg/L Rifampin in 5mL, in the YEP liquid nutrient medium of 50mg/L Streptomycin sulphate, 28 ℃, the 250rpm shaking culture is spent the night, get 1 μ L and carry out the PCR evaluation, the used upstream primer of PCR is NPTII-F:5 '-GTCACTGAAGCGGGAAGGG-3 ', downstream primer is NPTII-R:5 '-CGGCGATACCGTAAAGCAC-3 ', the PCR reaction system is: contain in the PCR reaction mixture of 20 μ L: 2.0 μ L, 10 * PCR buffer, 1.6 μ L 2.5mM dNTP mixture, 1.0 the Agrobacterium bacterium liquid that μ L is to be detected, 0.5 μ L 10mM NPTII-F primer 0.5 μ L 10mM NPTII-R primer, 13.9 μ L ddH ₂O.The PCR reaction conditions is: 94 ℃ 10 minutes; 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 1 minute, 30 circulations; 72 ℃ 7 minutes.Positive bacteria is come to the specific band of 493bp, positive bacterium colony called after EHA105/pCAMBIA2301.

The preparation of reorganization bacterium LBA4404/pCAMBIA2301 is identical with preparation and the method for transformation of above-mentioned EHA105/pCAMBIA2301 with method for transformation, that different is soil Agrobacterium LBA4404 (Ooms G, Hooykaas PJJ, van Veen RJM, van Beelen P, Regensburg-Tuink AJG, Schi lperoort RA (1982) Octopine Ti-plasmiddeletion mutants of Agrobacterium tumefaciens with emphasis on the right sideof the T-region.Plasmid 7:15-29, the public can obtain from Institute of Botany, Chinese Academy of Sciences.) replacing EHA105, bacterium LBA4404/pCAMBIA2301 obtains recombinating.

2) contain the preparation of the reorganization Agrobacterium solution of foreign gene

28 ℃ of shaking culture 24h in being seeded in the above-mentioned reorganization bacterium EHA105/pCAMBIA2301 that obtains in the YEP substratum that contains 50mg/L Rifampin, 50mg/L kantlex and 50mg/L Streptomycin sulphate, to increased logarithmic phase (the about 0.6-0.8 of OD600), the bacterium liquid of the bacterium EHA105/pCAMBIA2301 that recombinates this moment is orange can be used for transforming.

The bacterium liquid of the reorganization bacterium EHA105/pCAMBIA2301 of the about 0.6-0.8 of OD600 is put into the 30mL centrifuge tube, the centrifugal 10min of 3725rpm, abandon supernatant, thalline at the bottom of the collection tube, be total to culture medium 2 resuspended thalline with common culture medium 1 of liquid and liquid respectively, with bacterial concentration transfer to the OD value be 0.5,25 ℃ leave standstill behind the 1h standby, the reorganization Agrobacterium solution 2 that obtains containing the reorganization Agrobacterium solution 1 of foreign gene respectively and contain foreign gene.

The prescription of the common culture medium 1 of liquid is as follows: the 1/2MS minimum medium, 3.9g/L 2-morphine acid-ethyl sulfonic acid, 0.2g/L Sulfothiorine, 400mg/L L-halfcystine, 0.154g/L dithiothreitol (DTT), 200 μ mol/L Syringylethanones, the 4mg/L 6-benzyladenine, 30,000mg/L sucrose, pH5.6.

The prescription of the prescription of the common culture medium 2 of liquid and the common culture medium 1 of liquid is basic identical, and different is that the common culture medium 2 of liquid comprises the 5mg/L Silver Nitrate.

3) infect

50 immersed respectively in the reorganization Agrobacterium solution that above-mentioned two kinds of obtaining respectively of 30mL contain foreign gene by the 1 acceptor soybean hypocotyl that obtains infect, during constantly vibration, time of infection is 1h;

4) cultivate altogether

To take out through the soybean hypocotyl after infecting respectively, on aseptic filter paper, blot bacterium liquid, be placed on the common culture medium (add 6, the liquid of 000mg/L agar is culture medium altogether) that is covered with filter paper, 4d under 25 ℃ of shading conditions obtains common cultivation back soybean hypocotyl respectively.

After cultivating altogether soybean hypocotyl is used distilled water flushing 3 times successively, be total to culture medium flushing 4 times with containing Pyocianil and Reflin liquid.

5) bud inducing culture: respectively the explant after the above-mentioned common cultivation that obtains is put into bud inducing culture 2 respectively and carry out the bud inducing culture:

Bud inducing culture 2 prescriptions are as follows: 1/2MS minimum medium, 0.2mg/L indolebutyric acid, 30,000mg/L sucrose, 300mg/L Pyocianil, 300mg/L Reflin, 8,000mg/L agar, 4mg/L 6-benzyladenine, 5mg/L Silver Nitrate.

6) screening and culturing

Hypocotyl after the bud that above-mentioned cultivation is obtained is induced transfer to screening culture medium (add the 100mg/L kantlex in the bud inducing culture 2, pH5.8) on, subculture once obtains indefinite bud (this moment, indefinite bud length was 3cm) after cultivating for 4 weeks weekly.The temperature 25-28 of described screening and culturing ℃, intensity of illumination is 70 μ Em ^-2S ^-1, photoperiod control 18/6h (light/dark);

7) regeneration plant takes root and transplanting

When treating that indefinite bud length is 3cm (hypocotyl was cultivated for 4 weeks on screening and culturing), the base portion of being close to indefinite bud downcuts it, with the step that the otch of the indefinite bud that downcuts immerses the 2mg/L indolebutyric acid aqueous solution, carries out root culture.Obtain having the seedling of the adventive root of 2cm, seedling is taken out, water washes away the substratum that root adheres to, be transplanted to (turfy soil and vermiculite 1: 1 in the composite soil then, mass ratio), preserving moisture to cultivate changes the greenhouse over to after 1 week and carries out normal cultivation management, and pCAMBIA2301 regeneration soybean plant strain (have add the Silver Nitrate processing in the EHA105 strain cultures of pCAMBIA2301 plasmid obtain soybean plant strain) is changeed in the contrast that obtains the commentaries on classics pCAMBIA2301 regeneration soybean plant strain (have do not add in the EHA105 strain cultures of pCAMBIA2301 plasmid Silver Nitrate handle obtain soybean plant strain) of EHA105 mediation and EHA105 mediation respectively;

Adopting uses the same method carries out above-mentioned experiment with reorganization bacterium LBA4404/pCAMBIA2301, and pCAMBIA2301 regeneration soybean plant strain (have add the Silver Nitrate processing in the LBA4404 strain cultures of pCAMBIA2301 plasmid obtain soybean plant strain) is changeed in the contrast that obtains the commentaries on classics pCAMBIA2301 regeneration soybean plant strain (have do not add in the LBA4404 strain cultures of pCAMBIA2301 plasmid Silver Nitrate handle obtain soybean plant strain) of LBA4404 mediation and LBA4404 mediation respectively;

The prescription of root media is as follows: the 1/2MS minimum medium, and 20,000mg/L sucrose, the 150mg/L Reflin, the 10mg/L kantlex, 8,000mg/L agar is formed, and the pH value is 5.6.

The investigation gus gene is contaminated ability and hypocotyl replying Agrobacterium in the hope of determining different Agrobacteriums to soybean hypocotyl respectively at above-mentioned 4 kinds of regeneration soybean plant strain (hypocotyl) expressions that change pCAMBIA2301.

The expression frequency of statistics gus gene, the expression frequency of gus gene calculates by the following method: (the bud number by GUS histochemical stain test positive/obtain all resistant buds numbers of surviving on screening culture medium) * 100%.Described GUS histochemical stain is operated by the following method: bacterium liquid is infected the different common cultivation explant that obtains put into GUS staining fluid (the Jefferson R A that contains 1mM 5-bromo-4 chloro-3-indoles glucosides (X-gluc), KavanghT A and Bevan M W. (1987) GUS-fusions: β-glucuronidase as a sensitive andversatile gene fusion marker in higher plants.EMBO J 6:3901-3907, the public can obtain from Institute of Botany, Chinese Academy of Sciences).Open the pipe lid, be put in the vacuum-pumping container, vacuumize about 15min.Place 37 ℃ of insulation 12h.Dyeing time is decided according to the active power of GUS in the material.Decolour with 95% alcohol then.Take pictures under the stereoscopic microscope, present blue bud under the stereoscopic microscope and be accredited as positive bud, otherwise negative bud.The number of explant is 50, experiment triplicate, results averaged.

The result as shown in Figure 4, bar shaped post numbering from left to right is followed successively by 1,2,3,4 among the figure, wherein represents 4 kinds of different processing (the black post is represented the generation frequency of resistant buds, and white post is represented the expression frequency of gus gene) respectively.1 commentaries on classics pCAMBIA2301 regeneration soybean plant strain for the EHA105 mediation, 2 contrast commentaries on classics pCAMBIA2301 regeneration soybean plant strains for the EHA105 mediation, 3 commentaries on classics pCAMBIA2301 regeneration soybean plant strains for the LBA4404 mediation, 4 contrast commentaries on classics pCAMBIA2301 regeneration soybean plant strains for the LBA4404 mediation.

As can be seen from Figure 4, different strains is contaminated soybean hypocotyl, the final transformation efficiency that obtains exists difference, the expression frequency that pCAMBIA2301 regeneration soybean plant strain gus gene is changeed in the commentaries on classics pCAMBIA2301 regeneration soybean plant strain of LBA440 mediation and the contrast of LBA4404 mediation is respectively 9.6% and 6.2%, and the EHA105 mediation is changeed pCAMBIA2301 regeneration soybean plant strain and EHA105 mediation contrast and changeed the expression frequency of pCAMBIA2301 regeneration soybean plant strain gus gene and is respectively 20% and 10%.Simultaneously also as can be seen, adding Silver Nitrate in the Agrobacterium solution that contains foreign gene when infecting all has promoter action to the hypocotyl conversion that these two kinds of bacterial strains mediate, and the expression frequency of the gus gene of EHA105 mediation is higher than the LBA4404 mediation.

The influence that B, Agrobacterium concentration transform soybean hypocotyl

1, the acquisition of acceptor soybean hypocotyl

Test materials: identical with above-mentioned one;

Soybean seeds surface sterilization: identical with above-mentioned one;

Acceptor soybean hypocotyl: identical with above-mentioned one;

2, Agrobacterium preparation and conversion:

1) Agrobacterium preparation: identical with above-mentioned A method.

2) contain the preparation of the reorganization Agrobacterium solution of foreign gene: basic identical with the method for A, different is with reorganization bacterium EHA105/pCAMBIA2301 with liquid altogether culture medium 2 (containing the 5mg/L Silver Nitrate) dilute respectively for OD600 be 0.8,0.5,0.3 and 0.1.

3) infect: adopt the method for above-mentioned A, contaminate soybean hypocotyl explant 1h respectively after;

4) cultivate altogether: the method that adopts above-mentioned A; After cultivating 3 days altogether, obtain common cultivation explant respectively, detect the top meristematic zone GUS histochemical stain situation of cultivating explant altogether then, the transformation efficiency of statistics gene.

The transformation efficiency of gene calculates by the following method: (producing the explant number of the explant number of transgenic positive bud/be used to infect)) * 100.Described transgenic positive bud is meant the bud by GUS histochemical stain test positive; Described GUS histochemical stain is identical with above-mentioned A.Take pictures under the stereoscopic microscope, present blue bud under the stereoscopic microscope and be accredited as positive bud, otherwise negative bud.

The experiment triplicate, results averaged.

Statistics is as follows:

OD600 is that 0.8 transformation efficiency that infects the common cultivation explant that obtains is 16.1%;

OD600 is that 0.5 transformation efficiency that infects the common cultivation explant that obtains is 25.3%;

OD600 is that 0.3 transformation efficiency that infects the common cultivation explant that obtains is 13.9%;

OD600 is that 0.1 transformation efficiency that infects the common cultivation explant that obtains is 4.9%;

Map as shown in Figure 5, the bar shaped post from left to right is numbered 1,2,3,4 respectively among the figure, and the dilution of representative reorganization Agrobacterium is 0.8,0.5,0.3 and 0.1 processing for OD600 respectively.As seen from the figure, along with the dilution of bacterial concentration, transformation efficiency reduces significantly, and OD600 is 0.5 o'clock, and transformation efficiency is the highest.When to contaminate hypocotylar Agrobacterium concentration be (OD600) 0.8, hypocotyl apical meristem very short time on substratum had altogether had a strong impact on the regeneration of later stage indefinite bud with regard to brownization necrosis.

The influence that C, immerged time transform soybean hypocotyl

1, the acquisition of acceptor soybean hypocotyl

Test materials: identical with above-mentioned one;

Soybean seeds surface sterilization: identical with above-mentioned one;

Acceptor soybean hypocotyl: identical with above-mentioned one;

2, Agrobacterium preparation and conversion:

1) Agrobacterium preparation: identical with above-mentioned A method,

2) contain the preparation of the reorganization Agrobacterium solution of foreign gene: contain the preparation of the reorganization Agrobacterium solution of foreign gene: basic identical with the method for A, different is with reorganization bacterium EHA105/pCAMBIA2301 with liquid altogether culture medium 2 (containing the 5mg/L Silver Nitrate) dilution be 0.5 for OD600.

3) infect: adopt the method for above-mentioned A, different is that time of infection is 0.5h, 1h, 1.5h, 2h, 3h, 4h, 5h, 6h, 7h, 8h;

4) cultivate altogether: the method that adopts above-mentioned A; Obtain different common cultivation explants.

Detect GUS because of the transient expression frequency with above-mentioned A method.

The number of explant is 30, experiment triplicate, results averaged.

The result is as follows:

Time of infection is 0.5h, and gus gene transient expression frequency is 12%,

Time of infection is 1h, and GUS is 12.5% because of the transient expression frequency,

Time of infection is 1.5h, and gus gene transient expression frequency is 16.6%,

Time of infection is 2h, and gus gene transient expression frequency is 42.3%,

Time of infection is 3h, and gus gene transient expression frequency is 53.6%,

Time of infection is 4h, and gus gene transient expression frequency is 73%,

Time of infection is 5h, and gus gene transient expression frequency is 53.8%,

Time of infection is 6h, and gus gene transient expression frequency is 37.5%.

Map as shown in Figure 6, bar shaped post numbering from left to right is followed successively by 1,2,3,4,5,6,7,8 among the figure.1 is dip-dye 0.5h, and 2 for contaminating 1h, and 3 for contaminating 1.5h, and 4,5,6,7,8 are respectively dip- dye 2,3,4,5,6h.

As seen from the figure, be that gus gene transient expression frequency is the highest when reaching 4h in 0.5 the bacterium liquid when explant immerses OD600, frequency reaches 73.0%.Along with the further prolongation of immerged time, explant is being prone to brownization of enzymatic on the culture medium altogether, thereby has reduced transformation efficiency.

D, altogether incubation time is to the influence of soybean hypocotyl gus gene transient expression frequency

1, the acquisition of acceptor soybean hypocotyl

Test materials: identical with above-mentioned one;

Soybean seeds surface sterilization: identical with above-mentioned one;

Acceptor soybean hypocotyl: identical with above-mentioned one;

2, Agrobacterium preparation and conversion:

1) Agrobacterium preparation: identical with above-mentioned A method;

2) contain the preparation of the reorganization Agrobacterium solution of foreign gene: identical with the method for A.

3) infect: adopt the method for above-mentioned A, different is with OD600 among the A is that 0.5 bacterium liquid EHA105/pCAMBIA2301 infects, and time of infection is 4h;

4) cultivate altogether: the method that adopts above-mentioned A; That different is common cultivation 2d, 3d, and 4d obtains different common cultivation explants behind the 5d.

The detection of transient expression frequency: the gus gene transformation efficiency calculates by the following method: (producing the explant number of the explant number of transgenic positive bud/be used to infect)) * 100.Described transgenic positive bud is meant the bud by GUS histochemical stain test positive; Described GUS histochemical stain is identical with above-mentioned A method.Take pictures under the stereoscopic microscope, present blue bud under the stereoscopic microscope and be accredited as positive bud, otherwise negative bud.

The number of explant is 30, experiment triplicate, results averaged.

The result is as follows:

Cultivating the common cultivation explant gus gene transient expression frequency that 2d obtains altogether is 50.4%;

Cultivating the common cultivation explant gus gene transient expression frequency that 3d obtains altogether is 81.4%;

Cultivating the common cultivation explant gus gene transient expression frequency that 4d obtains altogether is 79.1%;

Cultivating the common cultivation explant gus gene transient expression frequency that 5d obtains altogether is 76.3%.

The mapping as shown in Figure 7, as seen from the figure, when 3d, gus gene transient expression rate rises significantly, cultivate altogether 4 and 5d after, gus gene transient expression frequency is similar with 3d.In addition, cultivate 5d altogether after, explant begins significantly brownization even rotten soft, is unfavorable for evoking adventive bud and even final Cheng Miao.Therefore, the suitableeest incubation time altogether is 3d.

E, antioxidant are to the influence of gus gene transient expression in soybean hypocotyl

1, the acquisition of acceptor soybean hypocotyl

Test materials: identical with above-mentioned one;

Soybean seeds surface sterilization: identical with above-mentioned one;

Acceptor soybean hypocotyl: identical with above-mentioned one;

2, Agrobacterium preparation and conversion:

1) Agrobacterium preparation: identical with above-mentioned A method.

2) contain the preparation of the reorganization Agrobacterium solution of foreign gene: identical with the method for A, different is respectively in liquid culture medium 2 and the liquid that do not add antioxidant (L-halfcystine, dithiothreitol (DTT) and Sulfothiorine) cultivation in the culture medium (not adding antioxidant L-halfcystine, dithiothreitol (DTT) and Sulfothiorine in the culture medium 2 altogether at liquid) altogether altogether;

3) infect: adopt the method for above-mentioned A, different is with OD600 among the A is that 0.5 bacterium liquid EHA105/pCAMBIA2301 infects, and time of infection is 4h;

4) cultivate altogether: the method that adopts above-mentioned A; Cultivate 3d altogether, different is is cultivating used substratum altogether and is not adding in the used substratum of common cultivation of antioxidant (L-halfcystine, dithiothreitol (DTT) and Sulfothiorine) and cultivate respectively; Obtain testing common cultivation explant respectively and contrast and cultivate explant altogether.

The number of explant is 50, experiment triplicate, results averaged.

A: observations is as follows:

Browning appears in surface (Fig. 8 A) and cut sides (the left figure of Fig. 8 E) that explant is cultivated in contrast altogether, the apical meristem necrosis (Fig. 8 C, shown in the arrow, and the left figure of Fig. 8 E), be unfavorable for that evoking adventive bud is to final Cheng Miao.

Experiment is cultivated the explant browning altogether and is significantly reduced (Fig. 8 B); Apical meristem normal (Fig. 8 D), the brownization degree of reduction soybean hypocotyl otch (the right figure of Fig. 8 E, and the right figure of 8F).

B: will test to cultivate explant altogether and contrast and cultivate explant altogether by the GUS histochemical stain, statistics gus gene expression frequency.

The result is as follows: the expression frequency that the explant gus gene is cultivated in contrast altogether is 2.3%, and the expression frequency that the explant gus gene is cultivated in experiment altogether is 62%.

From The above results as can be seen, antioxidant is handled and can be reduced brownization and strengthen gus gene in hypocotyl meristematic tissue and vertical expression.When cultivating soybean hypocotyl in the substratum that does not add antioxidant, the transient expression rate of gus gene is only 2.3%, and soybean hypocotyl is cultivated in containing the antioxidant substratum, the transient expression frequency of gus gene is increased to 62%, has increased by 27 times than the former.Antioxidant can significantly reduce the brownization degree of soybean hypocotyl otch, illustrates that the raising of transient expression rate of gus gene and the brownization degree that antioxidant reduces the soybean hypocotyl otch have confidential relation.Therefore, in the transgenosis process of soybean, in substratum, add antioxidant and can improve genetic transformation efficiency.

The transient expression position of gus gene in the hypocotyl tissue studied, (Fig. 8 E is the influence of antioxidant to hypocotyl gus gene transient expression as Fig. 8 E and 8F, and wherein left side figure is the soybean hypocotyl of nonreactive oxidizer treatment, brownization of explant and do not detect the expression of gus gene.Right figure is that the hypocotyl explant apical meristem that gus gene is handled at antioxidant is expressed.Fig. 8 F is the expression of antioxidant enhancement soybean hypocotyl gus gene, and wherein left side figure is that hypocotyl is cultivated on the substratum of no antioxidant brownization of explant.Right figure is that the hypocotyl cultivation is containing the antioxidant substratum, and gus gene is at the hypocotyl strong expression.) shown in, the dyeing part major part is positioned at hypocotyl vascular bundle form layers tissue.These presentation of results, antioxidant have mainly strengthened Agrobacterium to the histiocytic dip-dye of hypocotylar vascular bundle form layers.Hypocotylar vascular bundle cambial cell has very strong meristematic capacity, can break up rapidly, and this has vital role to improving the soybean gene transformation frequency.

F, AS concentration are to the influence of gus gene transient expression frequency

1, the acquisition of acceptor soybean hypocotyl

Test materials: identical with above-mentioned one;

Soybean seeds surface sterilization: identical with above-mentioned one;

Acceptor soybean hypocotyl: identical with above-mentioned one;

2, Agrobacterium preparation and conversion:

1) Agrobacterium preparation: identical with above-mentioned A method;

2) contain the preparation of the reorganization Agrobacterium solution of foreign gene: basic identical with the method for A, different is to add the different Syringylethanones of measuring (AS) in the culture medium 2 altogether at liquid, make Syringylethanone (AS) be respectively 0 μ M in the concentration that liquid is total in the culture medium 2,50 μ M, 100 μ M, 150 μ M, 200 μ M, 300 μ M and 400 μ M, middle cultivation;

3) infect: the method for A in the employing, different is with OD600 among the B is that 0.5 bacterium liquid EHA105/pCAMBIA2301 infects, time of infection is 4h;

4) cultivate altogether: the method that adopts above-mentioned A; Cultivated for 4 weeks altogether, cultivating used culture medium culturing altogether; Obtain common cultivation explant 0,1,2,3,4,5,6.Every processing explant number is 20, experiment triplicate, results averaged.

The transformation efficiency of gus gene calculates by the following method: (producing the explant number of the explant number of transgenic positive bud/be used to infect)) * 100.Described transgenic positive bud is meant the bud by GUS histochemical stain test positive; Described GUS histochemical stain is identical with above-mentioned A method.Take pictures under the stereoscopic microscope, present blue bud under the stereoscopic microscope and be accredited as positive bud, otherwise negative bud.

Result's demonstration,

Cultivating explant 0 (0 μ M) gus gene transient expression frequency altogether is 21%;

Cultivating explant 1 (50 μ M) gus gene transient expression frequency altogether is 52.3%;

Cultivating explant 2 (100 μ M) gus gene transient expression frequency altogether is 71.2%;

Cultivating explant 3 (150 μ M) gus gene transient expression frequency altogether is 77.8%;

Cultivating explant 4 (200 μ M) gus gene transient expression frequency altogether is 83%;

Cultivating explant 5 (300 μ M) gus gene transient expression frequency altogether is 81.3%;

Cultivating explant 6 (400 μ M) gus gene transient expression frequency altogether is 78.6%;

Map as shown in Figure 9, the bar shaped post is numbered from left to right and is followed successively by 1,2 among the figure, and 3,4,5,6,7, representing Syringylethanone (AS) respectively in the culture medium is 0 μ M altogether at liquid, 50 μ M, 100 μ M, 150 μ M, 200 μ M, 7 kinds of processing that 300 μ M and 400 μ M carry out.As seen from the figure, cultivation stage adds raising and the increase gus gene transient expression frequency that 200 μ M Syringylethanones help transformation frequency altogether.

Embodiment 2: change pCAMBIA2301 plasmid soybean plant strain and gus gene in hypocotylar expression

One, changes the acquisition of pCAMBIA2301 soybean plant strain

Method one:

1, the acquisition of acceptor soybean hypocotyl

1) test materials: soybean (Glycine max) kind: black farming 44

2) soybean seeds surface sterilization: select anosis, full soybean seeds and be tiled in the uncovered culture dish, put into built-in-fill the moisture eliminator of 100mL clorox stoste beaker, slowly add the 4mL concentrated hydrochloric acid along beaker then, the close drying device places the stink cupboard 18h that sterilizes.Seed after the sterilization is placed in the germination medium (500mg/L agar, pH 5.8 for 1/2MS minimum medium+15,000mg/L sucrose+7), places 25 ℃, 120 μ Em ^-2S ^-1Cultivate 5d under the condition of light intensity (periodicity of illumination 18/6h).

3) preparation of explant

Soybean hypocotyl: when cultivating 5d, sprout soybean peel this moment and break, primary leaf (primary leaves) does not also expose cotyledon.In Bechtop, take out soybean, carefully remove kind of a skin, cotyledon is peeled off.With scalpel terminal bud is excised then, the hypocotyl that cuts the 1cm length with apical meristem is as the acceptor soybean hypocotyl.

2, Agrobacterium preparation and conversion

1) Agrobacterium preparation

The competent preparation of Agrobacterium: the single colony inoculation of picking soil Agrobacterium EHA105 contains the 50mg/L Rifampin in 5mL, the YEP liquid nutrient medium of 50mg/L Streptomycin sulphate (contains 10, the 000mg/L Tryptones, 10, the 000mg/L yeast extract, 5,000mg/L sodium-chlor, pH7.0) in, 28 ℃, 250rpm shaking culture spend the night; Be inoculated in 40mL by 1: 100 and contain the 50mg/L Rifampin, continue to cultivate 4-6h in the YEP liquid nutrient medium of 50mg/L Streptomycin sulphate; 4 ℃, 5, the centrifugal 10min of 000rpm removes supernatant; CaCL with 600 μ L ice precooling 0.05mol/L ₂The solution washing thalline; 4 ℃, 5, the centrifugal 10min of 000rpm removes supernatant; CaCL with 200 μ L ice precooling 0.05mol/L ₂Resuspended thalline is in 4 ℃ of preservations.

Plasmid is to the conversion of Agrobacterium: add pCAMBIA2301 (www.cambia.org.au) plasmid DNA (this plasmid contains selectable marker gene NPTII and gus reporter gene) of about 0.5 μ g in the competence Agrobacterium, mixing is placed 5min on ice gently; Place liquid nitrogen 3min then; Be transferred to 37 ℃ of incubation 5min rapidly; Add 500 μ L YEP liquid nutrient mediums, 28 ℃ of shaking culture 5-6h; 5, the centrifugal 3min of 000rpm removes most of supernatant, surplus 200 μ L, suspension thalline; Evenly coat the YEP that contains 50mg/L Rifampin, 50mg/L Streptomycin sulphate and 50mg/L kantlex and select on the flat board, be inverted for 28 ℃ and cultivated two days, obtain single bacterium colony.Picking list colony inoculation contains the 50mg/L Rifampin in 5mL, in the YEP liquid nutrient medium of 50mg/L Streptomycin sulphate, 28 ℃, the 250rpm shaking culture is spent the night, get 1 μ L and carry out the PCR evaluation, the used upstream primer of PCR is NPTII-F:5 '-GTCACTGAAGCGGGAAGGG-3 ', downstream primer is NPTII-R:5 '-CGGCGATACCGTAAAGCAC-3 ', the PCR reaction system is: contain in the PCR reaction mixture of 20 μ l: 2.0 μ L, 10 * PCR buffer, 1.6 μ L 2.5mM dNTP mixture, 1.0 the Agrobacterium bacterium liquid that μ L is to be detected, 0.5 μ L 10mM NPTII-F primer 0.5 μ L 10mM NPTII-R primer, 13.9 μ L ddH ₂O.The PCR reaction conditions is: 94 ℃ 10 minutes; 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 1 minute, 30 circulations; 72 ℃ 7 minutes.Positive bacteria is come to the specific band of 493bp, positive bacterium colony called after EHA105/pCAMBIA2301.

2) contain the preparation of the reorganization Agrobacterium solution of foreign gene

The above-mentioned reorganization bacterium EHA105/pCAMBIA2301 that obtains is seeded in 28 ℃ of shaking culture 24h in the YEP substratum that contains 50mg/L Rifampin, 50mg/L kantlex and 50mg/L Streptomycin sulphate, to increased logarithmic phase (the about 0.6-0.8 of OD600), the bacterium liquid of the bacterium EHA105/pCAMBIA2301 that recombinates this moment is orange can be used for transforming.

The bacterium liquid of the reorganization bacterium EHA105/pCAMBIA2301 of OD6000.8 is put into the 30mL centrifuge tube, the centrifugal 10min of 3725rpm, abandon supernatant, thalline at the bottom of the collection tube is total to culture medium (1/2MS minimum medium, 3.9g/L2-morphine acid-ethyl sulfonic acid with liquid, 0.2g/L Sulfothiorine, 400mg/L L-halfcystine, 0.154g/L dithiothreitol (DTT), 200 μ mol/L Syringylethanones, the 4mg/L 6-benzyladenine, the 5mg/L Silver Nitrate, 30,000mg/L sucrose, pH 5.6) resuspended thalline, bacterial concentration is transferred to the OD value be 0.5,25 ℃ and leave standstill behind the 1h standbyly, obtain containing the reorganization Agrobacterium solution of foreign gene.

3) infect

Will 50 immerse in the reorganization Agrobacterium solution that contain foreign gene of the above-mentioned acquisition of 30mL and infect by the 1 acceptor soybean hypocotyl that obtains, during constantly vibration, time of infection is 4h;

4) cultivate altogether

To take out through the soybean hypocotyl after infecting, on aseptic filter paper, blot bacterium liquid, be placed on the common culture medium (add 6, the liquid of 000mg/L agar is culture medium altogether) that is covered with filter paper, 3d under 25 ℃ of shading conditions obtains common cultivation back soybean hypocotyl.

After cultivating altogether soybean hypocotyl is used distilled water flushing 3 times successively, be total to culture medium flushing 4 times with containing Pyocianil and Reflin liquid.

5) bud inducing culture

Soybean hypocotyl after will washing is then transferred to bud inducing culture (1/2MS minimum medium, 4mg/L 6 benzyladenines, 0.2mg/L indolebutyric acid, 30,000mg/L sucrose, 300mg/L Pyocianil, 300mg/L Reflin, 8,000mg/L agar, the 5mg/L Silver Nitrate, pH 5.8) go up and recover to cultivate, culture temperature is 27 ℃, and intensity of illumination is 70 μ Em ^-2S ^-1, photoperiod control 18/6h cultivates 6d, obtains inducing the back hypocotyl.

6) screening and culturing

After above-mentioned recovery cultivated induce the back hypocotyl transfer to screening culture medium (the bud inducing culture adds the 100mg/L kantlex, pH5.8) on, subculture once obtains indefinite bud (this moment, indefinite bud length was 2.5cm) after cultivating for 4 weeks weekly.27 ℃ of the temperature of described screening and culturing, intensity of illumination are 70 μ Em ^-2S ^-1, photoperiod control 18/6h light/dark.

7) regeneration plant takes root and transplanting

When treating the indefinite bud elongation for 2.5cm, the base portion of being close to indefinite bud downcuts it, the base portion (being otch) of the indefinite bud that downcuts immersed in the 2mg/L indolebutyric acid aqueous solution soak 5min, change root media (1/2MS minimum medium over to after blotting with aseptic filter paper then, 20,000mg/L sucrose, the 150mg/L Reflin, the 10mg/L kantlex, 0.8% agar, the pH value is 5.6,27 ℃, cultivated 15 days, and obtained having the seedling of the adventive root of 2cm, seedling is taken out, water washes away the substratum that root adheres to, be transplanted to then (turfy soil and vermiculite mass ratio 1: 1) in the composite soil, change the greenhouse over to after 1 week of cultivation of preserving moisture and carry out normal cultivation management, obtain the regeneration soybean plant strain (hypocotyl) that pCAMBIA2301 is changeed in 20 strains.

Method two,

1, the acquisition of acceptor soybean hypocotyl

1) test materials: identical with method one;

2) soybean seeds surface sterilization: basic identical with method one, different is to cultivate 6d down at 22 ℃;

3) preparation of explant: basic identical with method one, different is to cut the hypocotyl of the 0.5cm length with apical meristem as the acceptor soybean hypocotyl;

2, Agrobacterium preparation and conversion

1) Agrobacterium preparation: identical with method one;

2) contain the preparation of the reorganization Agrobacterium solution of foreign gene: identical with method one;

3) infect: identical with method one;

4) cultivate altogether: basic identical with method one, different is to cultivate down at 22 ℃;

5) bud inducing culture: basic identical with method one, different is to cultivate down at 25 ℃;

6) screening and culturing: basic identical with method one, different is to cultivate down at 25 ℃;

7) the taking root and transplant of regeneration plant: basic identical with method one, different is that the base portion of being close to indefinite bud downcuts it when treating the indefinite bud elongation for 3cm, the base portion (being otch) of the indefinite bud that downcuts is immersed in the 2mg/L indolebutyric acid aqueous solution soak 2min; And 25 ℃ of seedling of cultivating down the adventive root that obtains having 3cm.

Method three,

1, the acquisition of acceptor soybean hypocotyl

1) test materials: identical with method one;

2) soybean seeds surface sterilization: basic identical with method one, different is to cultivate 6d down at 23 ℃;

3) preparation of explant: basic identical with method one, different is to cut the hypocotyl of the 0.8cm length with apical meristem as the acceptor soybean hypocotyl;

2, Agrobacterium preparation and conversion

1) Agrobacterium preparation: identical with method one;

2) contain the preparation of the reorganization Agrobacterium solution of foreign gene: identical with method one;

3) infect: identical with method one;

4) cultivate altogether: basic identical with method one, different is to cultivate down at 23 ℃;

5) bud inducing culture: basic identical with method one, different is to cultivate down at 28 ℃;

6) screening and culturing: basic identical with method one, different is to cultivate down at 28 ℃;

7) the taking root and transplant of regeneration plant: basic identical with method one, different is that the base portion of being close to indefinite bud downcuts it when treating the indefinite bud elongation for 4cm, the base portion (being otch) of the indefinite bud that downcuts is immersed in the 2mg/L indolebutyric acid aqueous solution soak 3min; And 28 ℃ of seedling of cultivating down the adventive root that obtains having 4cm.

Two, gus gene is in hypocotylar expression

The expression of the gus gene in the hypocotyl that detection method one obtains.

The number of explant is 50, below tests equal triplicate, results averaged.

Observe the expression of the gus gene in each stage with the GUS histochemical stain, Figure 10 is the part physical map (containing gus gene) of plasmid pCAMBIA2301.

Specific as follows:

Cultivate altogether and finish to found that two days later to explant GUS histochemical stain, gus gene is at explant top strong expression, and the expression position mainly concentrates on the meristematic tissue (Figure 11 A) around leader (the cut during the preparation explant) base portion.

Screening and culturing to explant GUS histochemical stain, found that after one week, and the meristematic tissue of expressing gus gene begins propagation (Figure 11 B) and along with the prolongation of incubation time is progressively induced and formed bud original hase (Figure 11 C).These bud original hases are differentiated to form indefinite bud, and from Figure 11 D as can be seen, the soybean hypocotyl apical meristem is differentiated to form the bud original hase and expresses gus gene, is distributed in the form of a ring around the leader base portion.In contrast, leader residue tissue and face tissue are not dyeed by GUS, and it is mitogenetic and accept the ability of foreign DNA that the meristematic tissue that the leader base portion is described has intensive, is " the target cell tissue " in the Agrobacterium-mediated Transformation hypocotyl process.

At the hypocotyl high frequency regeneration indefinite bud in two weeks of screening and culturing, arrow is depicted as GUS male regenerated adventitious bud, and the non-transformed cell around it is brownization dead (Figure 11 E) progressively.The hypocotyl explant that transforms was cultivated for 4 weeks on screening culture medium, and indefinite bud can be stretched to 3cm, can be transferred to root media.

(the right figure of Figure 12 B is the dyeing of ripe leaflet to change indefinite bud, blade, the seed of pCAMBIA2301 plasmid soybean plant strain by further GUS histochemical stain, Figure 12 C is that dyeing, Figure 12 D of the ripe leaflet of different transgenic lines is the dyeing of immature seed), transfer-gen plant is screened (Figure 12), and the result is:

The GUS stained positive of changeing pCAMBIA2301 plasmid soybean plant strain indefinite bud is 20 strains (Figure 12 A);

The GUS stained positive of changeing the blade of pCAMBIA2301 plasmid soybean plant strain is 20 strains (Figure 12 B, 12C);

The GUS stained positive of changeing the seed of pCAMBIA2301 plasmid soybean plant strain is 20 strains (Figure 12 D);

Therefore, obtaining 20 strains altogether changes the positive soybean plant strain of pCAMBIA2301 plasmid soybean, and can educate.

Adopt the expression of the gus gene in the hypocotyl that use the same method detection method two and method three obtain, result and method one no significant difference.

Claims (10)

1. a soybean heredity method for transformation comprises the steps:

1) uses the reorganization Agrobacterium solution that contains foreign gene to infect the hypocotyl of soybean, obtain infecting the back hypocotyl;

2) the back hypocotyl of infecting that step 1) is obtained carries out common cultivation and obtains common cultivations hypocotyl afterwards;

3) with step 2) obtain cultivate the back hypocotyl and carry out hypocotyl after the bud inducing culture obtains bud and induces altogether;

4) hypocotyl after the bud that step 3) is obtained is induced carries out the hypocotyl that screening and culturing obtains screening the back survival;

5) take off indefinite bud the hypocotyl that after the screening that step 4) obtains, survives, indefinite bud is carried out root culture, obtain the genetically engineered soybean seedling.

2. method according to claim 1 is characterized in that:

The hypocotyl of described soybean is the long hypocotyl of 0.5cm-1cm with apical meristem, and the hypocotyl of described soybean is specially 0.5cm, 0.8cm or the long hypocotyl of 1cm with apical meristem;

The hypocotyl of described acceptor soybean is prepared as follows: soybean seeds was sprouted 5 days-6 days, removed kind of a skin, cotyledon, terminal bud, obtained having the long hypocotyl of 0.5cm-1cm of apical meristem;

The hypocotyl of described acceptor soybean specifically is prepared as follows: soybean seeds was sprouted 5 days or 6 days, removed kind of a skin, cotyledon, terminal bud, obtained having 0.5cm, 0.8cm or the long hypocotyl of 1cm of apical meristem.

3. method according to claim 1 and 2 is characterized in that:

In the step 1), the described reorganization Agrobacterium solution that contains foreign gene is for to be prepared as follows: the described reorganization Agrobacterium that contains foreign gene is resuspended in liquid altogether in the culture medium, transfer to the OD value and be 0.3-0.8,25 ℃ leave standstill 1h, and Agrobacterium solution obtains recombinating; Described OD value is specially 0.3,0.4,0.5,0.6 or 0.8;

Described liquid culture medium altogether is prepared as follows: add 2-morphine acid-ethyl sulfonic acid in the 1/2MS substratum, Sulfothiorine, the L-halfcystine, dithiothreitol (DTT), Syringylethanone, 6-benzyladenine, Silver Nitrate and sucrose, obtain described liquid culture medium altogether, described 2-morphine acid-ethyl sulfonic acid is 3.9g/L in the concentration that described liquid is total in the culture medium, described Sulfothiorine is 0.2g/L in the concentration that described liquid is total in the culture medium, described L-halfcystine is 400mg/L in the concentration that described liquid is total in the culture medium, described dithiothreitol (DTT) is 0.154g/L in the concentration that described liquid is total in the culture medium, described Syringylethanone is 200 μ mol/L in the concentration that described liquid is total in the culture medium, described 6-benzyladenine is 4mg/L in the concentration that described liquid is total in the culture medium, described Silver Nitrate is 5mg/L in the concentration that described liquid is total in the culture medium, described sucrose is 30 in the concentration that described liquid is total in the culture medium, 000mg/L, described the liquid pH of culture medium altogether are 5.6;

Step 2) in, describedly cultivate used substratum altogether and be prepared as follows: add agar in the culture medium altogether to described liquid and obtain the used substratum of common cultivation, described agar is 6 in described concentration of cultivating altogether in the used substratum, 000mg/L, the described pH that cultivates used substratum altogether is 5.6;

In the step 3), the used substratum of described bud inducing culture is prepared as follows: add 6-benzyladenine in the 1/2MS substratum, indolebutyric acid, sucrose, Pyocianil, Reflin, Silver Nitrate and agar, obtain the used substratum of described bud inducing culture, the concentration of described 6-benzyladenine in the used substratum of described bud inducing culture is 4mg/L, the concentration of described indolebutyric acid in the used substratum of described bud inducing culture is 0.2mg/L, the concentration of described sucrose in the used substratum of described bud inducing culture is 30,000mg/L, the concentration of described Pyocianil in the used substratum of described bud inducing culture is 300mg/L, the concentration of described Reflin in the used substratum of described bud inducing culture is 300mg/L, the concentration of described Silver Nitrate in the used substratum of described bud inducing culture is 5mg/L, the concentration of described agar in the used substratum of described bud inducing culture is 8,000mg/L, the pH of the used substratum of described bud inducing culture is 5.8;

In the step 4), the used substratum of described screening and culturing is prepared as follows: add kantlex in the used substratum of described bud inducing culture, obtain the used substratum of described screening and culturing, the concentration of described kantlex in the used substratum of described screening and culturing is 100mg/L, and the pH of the used substratum of described screening and culturing is 5.8;

In the step 5), the used substratum of described root culture is prepared as follows: add sucrose in the 1/2MS substratum, Reflin, kantlex and agar, obtain the used substratum of described root culture, the concentration of described sucrose in the used substratum of described root culture is 20,000mg/L, the concentration of described Reflin in the used substratum of described root culture is 150mg/L, the concentration of described kantlex in the used substratum of described root culture is 10mg/L, the concentration of described agar in the used substratum of described root culture is 8,000mg/L, the pH value of the used substratum of described root culture is 5.6.

4. according to arbitrary described method among the claim 1-3, it is characterized in that:

In the step 1), the described time of infecting is 0.5h-8h; The described mode that infects is: the hypocotyl of described acceptor soybean is immersed contained in the reorganization Agrobacterium solution of foreign gene; The described time of infecting is specially 0.5h, 1h, 1.5h, 2h, 3h, 4h, 5h, 6h, 7h or 8h;

Step 2) in, described temperature of cultivating altogether is 22 ℃-25 ℃, and described temperature of cultivating altogether is specially 22 ℃, 23 ℃ or 25 ℃, and described the cultivation altogether to dark cultivated, and the described time of cultivating altogether is 2 days-5 days; The described time of cultivating altogether was specially 2 days, 3 days, 4 days or 5 days;

In the step 3), 25 ℃-28 ℃ of the temperature of described bud inducing culture, the temperature of described bud inducing culture is specially 25 ℃, 27 ℃ or 28 ℃, and intensity of illumination is 70 μ Em ^-2S ^-1, photoperiod control 18/6h (light/dark), incubation time is 6 days;

In the step 4), 25 ℃-28 ℃ of the temperature of described screening and culturing, the temperature of described screening and culturing is specially 25 ℃, 27 ℃ or 28 ℃, and intensity of illumination is 70 μ Em ^-2S ^-1, photoperiod control 18/6h light/dark, and subculture is once weekly; The time of described screening and culturing was 4 weeks;

In the step 5), 25 ℃-28 ℃ of the temperature of described root culture, the temperature of described root culture is specially 25 ℃, 27 ℃ or 28 ℃, and intensity of illumination is 70 μ Em ^-2S ^-1, photoperiod control 18/6h light/dark, incubation time 15 days.

5. according to arbitrary described method among the claim 1-4, it is characterized in that:

In the step 5), the length of described indefinite bud is 2.5cm-4cm.

6. according to arbitrary described method among the claim 1-5, it is characterized in that:

In the step 5), the length of described indefinite bud is 2.5cm, 3cm or 4cm.

7. according to arbitrary described method among the claim 1-6, it is characterized in that:

In step 2) after, before the step 3), also comprise successively with distilled water and contain Pyocianil and the Reflin liquid described hypocotylar step in back of cultivating altogether of culture medium flushing altogether.

In the described step 5), the described indefinite bud that takes off downcuts indefinite bud for the base portion from indefinite bud; Described take off the step of indefinite bud after, described indefinite bud is carried out the root culture step before, comprise that also it is the step of soaking in the indolebutyric acid aqueous solution of 2mg/L that the shearing end that will downcut indefinite bud immerses concentration.

8. according to arbitrary described method among the claim 1-7, it is characterized in that:

Described successively with distilled water with contain Pyocianil and Reflin liquid the culture medium flushing is described altogether and cultivate altogether in the hypocotylar step in back, the described liquid that contains Pyocianil and Reflin is total to culture medium and is prepared as follows: add Pyocianil and Reflin in the culture medium altogether to described liquid, the liquid that contains Pyocianil and Reflin that obtains is culture medium altogether, described Pyocianil is 300mg/L in the concentration that the described liquid that contains Pyocianil and Reflin is total in the culture medium, and described Reflin is 300mg/L in the concentration that the described liquid that contains Pyocianil and Reflin is total in the culture medium;

It is in the step of soaking in the indolebutyric acid aqueous solution of 2mg/L that the described shearing end that will downcut indefinite bud immerses concentration, and described soak time is 2min-5min; Described soak time is specially 2min, 3min or 5min; The position of the described indefinite bud of described cutting-out is a base portion of being close to indefinite bud;

In the described method, after described step 5), also comprise with having of obtaining of root culture greater than the seedling replanting of the adventive root of 2cm in composite soil, cultivated for 1 week, obtain the step of genetically engineered soybean plant, described composite soil is made up of turfy soil and vermiculite, and the mass ratio of described turfy soil and vermiculite is 1: 1, the length of described adventive root is preferably 2cm-4cm, is specially 2cm, 3cm or 4cm.

9. according to arbitrary described method among the claim 1-8, it is characterized in that:

Among the hypocotyl preparation method of described soybean, the used substratum of described sprouting is prepared as follows: add sucrose and agar in the 1/2MS substratum, obtain described sprouting and cultivate used substratum, the concentration that described sucrose is cultivated in the used substratum in described sprouting is 15,000mg/L, the concentration that described agar is cultivated in the used substratum in described sprouting is 7, and 500mg/L, the pH of the substratum that described sprouting is used are 5.8;

The temperature that described sprouting is cultivated is 22 ℃-25 ℃, and the temperature that described sprouting is cultivated is specially 22 ℃, 23 ℃, 25 ℃, and intensity of illumination is 120 μ Em ^-2S ^-1, periodicity of illumination is 18h/6h illumination/dark.

10. according to arbitrary described method among the claim 1-9, it is characterized in that:

The described reorganization Agrobacterium that contains foreign gene is following 1) or 2):

1), carrier pCAMBIA2301 is imported the reorganization Agrobacterium that agrobacterium tumefaciens lba4404 obtains;

2), carrier pCAMBIA2301 is imported the reorganization Agrobacterium that agrobacterium tumefaciens EHA105 obtains.

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