CN101144091B - Method for obtaining switchgrass transgene plant - Google Patents

Abstract

The present invention discloses a method that a switchgrass transgenic plant is obtained by adopting agrobacterium-mediated transformation. When the genetic transformation of the switchgrass is operated, a seed which burgeons after being sterilized is used as a transformation acceptor, and the agrobacterium transformation and the co-culture are adopted; by screening on culture mediums of SMBP1, SMBP2 and SMBP3 in sequence, a resistant bud is obtained; the resistant bud is cultivated into a seedling in a rooting culture medium SMO; the seedling is transplanted to a greenhouse or the field to perform the detection of the molecular biological method, and a transgenic plant containing the exogenous aim is obtained by screening according to the molecular biological method and the target trait. The transgenic method of the switchgrass established by the present invention overcomes the disadvantage that the dependance of the agrobacterium-mediated transformation to the tissue culture condition is strong, widens the genotype range of the genetic transformation, and simplifies the transformation process, to provides a new approach for the genetic improvement of the switchgrass by adopting the transgenic technology.

Description

A kind of method that obtains switchgrass transgene plant

Technical field

The invention belongs to biological technical field, relate to the gene transformation of bioenergy plant switchgrass, further relate to a kind of agrobacterium-mediated transformation that adopts and transform the method that obtains switchgrass transgene plant.

Background technology

Utilizing transgenic technology that energy-source plant is carried out genetic improvement is the new breakthrough of modern genetic engineering technology in the agriculture field utilization.Switchgrass is important new bio energy-source plant, has become one of major objective of gene transformation.Wherein, adopt the resistance of transgenic technology enhancing switchgrass, improve photosynthetic efficiency, increase biological yield, reducing aspects such as content of lignin and raising alcohol yied has become present research focus and has shown good prospects for application.

Advantages such as agriculture bacillus mediated gene transformation system dna fragmentation low because of foreign DNA structural integrity, inheritance stability, the copy number that it has conversion, that transform is bigger have become the prefered method that present most of plant genetic transforms.In the switchgrass transgenic plant that report obtains, also adopt agriculture bacillus mediated method to finish in the world.

Similar to the genetic transformation of other plant, adopt agrobacterium-mediated transformation conversing switchgrass conducted also exist the tissue culture technique dependency strong, transformation period is long, exist genotype restriction (at present switchgrass transforms successfully only be indivedual genotype), shortcomings such as regenerable cell position and transformed competence colibacillus cell are inconsistent are being very limited it at present by the widespread usage on the switchgrass of the difficult a large amount of acquisition embryo callus of tissue culture technique.Explore a kind of advantage that can utilize agriculture bacillus mediated system, avoid again, will play very big promoter action the gene transformation research of switchgrass with the genetic transforming method of callus as transformation receptor.

Summary of the invention

Problem or deficiency at above-mentioned existing switchgrass transgenic technology existence, the objective of the invention is to, a kind of method that obtains switchgrass transgene plant is provided, this method is a transformation receptor with the seed of sprouting, adopt agrobacterium-mediated transformation to transform and obtain switchgrass transgene plant, to simplify the genetic transformation procedures of switchgrass, shorten the transformation period, widen the genotype scope of genetic transformation.

In order to realize above-mentioned task, the present invention adopts following technical scheme:

A kind of method that obtains switchgrass transgene plant is characterized in that, this method is a transformation receptor with the seed of sprouting, and adopts agrobacterium-mediated transformation to transform and obtains switchgrass transgene plant, specifically carries out as follows:

Step 1: the sterilization of seed

Get the mature seed of switchgrass, sterilize with the Losantin of 3% concentration, the dark cultivation 12 hours used 3% Losantin two-stage sterilization again under 4 ℃ of conditions;

Step 2: the sprouting of seed

Seed on aseptic filter paper, under 24 ℃ of conditions, secretly is cultured to seed and sprouts;

Step 3: Agrobacterium-mediated Transformation and common cultivation

The mono-clonal of picking Agrobacterium under 28 ℃ of conditions, was cultivated 1～2 day with 200rpm/ minute～250rpm/ minute in the LB substratum that contains kantlex and Rifampin, to agrobacterium strains concentration be OD ₆₀₀=0.8～1.0, described LB substratum is: the 10g tryptone, and the 5g yeast extract, 10g sodium-chlor, pH are 7.0;

In the LB substratum, add Syringylethanone and continue to cultivate 1 hour～2 hours, change over to then in the aseptic 50mL centrifuge tube, under the normal temperature, 3500rpm/ minute, collected Agrobacterium in centrifugal 15 minutes, with the liquid MBP substratum Agrobacterium that suspends again, regulating agrobacterium strains concentration is OD ₆₀₀=0.5, wherein, liquid MBP substratum is: the mixing synthetics of MS inorganic salt and VITAMIN: 4.43g/L, maltose: 30g/L, 6-benzylaminopurine: 3mg/L, pH=5.8;

Laterally rive along the seed middle part, discard the half granule seed that does not contain radicle, add the good Agrobacterium bacterium liquid that suspends, add 50 μ l 100mM Syringylethanones simultaneously, after vacuumizing 60～90 minutes on the vaccum bench, under the normal temperature, 50rpm/ minute～60rpm/ minute cultivation 60～90 minutes;

Discard whole Agrobacterium bacterium liquid, remove the residual bacterium liquid of seed-coat with the aseptic filter paper suction, then seed is changed over to substratum MBP, under 20 ℃～24 ℃ conditions, the dark cultivation 2～3 days, wherein, the MBP substratum is: mixing synthetics 4.43g/L, the maltose 30g/L of MS inorganic salt and VITAMIN, 6-benzylaminopurine 3mg/L, agar powder 7.5g/L, pH=5.8;

Step 4: the screening of resistance seedling

Change seed over to screening culture medium SMBP1 successively, SMBP2 and SMBP3 under 24 ℃ of conditions, successively 2～3 weeks of illumination cultivation, obtain the resistance seedling; Wherein, screening culture medium SMBP1 is: mixing synthetics 4.43g/L, the maltose 30g/L of MS inorganic salt and VITAMIN, 6-benzylaminopurine 3mg/L, Totomycin 100mg/L, cephamycin 250mg/L, agar powder 7.5g/L, pH=5.8; Screening culture medium SMBP2 is: mixing synthetics 4.43g/L, the maltose 30g/L of MS inorganic salt and VITAMIN, 6-benzylaminopurine 3mg/L, Totomycin 75mg/L, cephamycin 250mg/L, agar powder 7.5g/L, pH=5.8; Screening culture medium SMBP3 is: mixing synthetics 4.43g/L, the maltose 30g/L of MS inorganic salt and VITAMIN, 6-benzylaminopurine 3mg/L, Totomycin 50mg/L, cephamycin 250mg/L, agar powder 7.5g/L, pH=5.8;

Step 5: the root culture of resistance seedling

When resistant buds growth 1cm～2cm is high, change among the root media SMO, 28 ℃, illumination condition was cultivated for 3～4 weeks down, to the seedling plant height be more than the 5cm, wherein, root media SMO is: mixing synthetics 2.22g/L, the sucrose 8g/L of MS inorganic salt and VITAMIN, agar powder 7.0g/L, pH=5.8;

Step 6: transplant to the greenhouse or the land for growing field crops is carried out molecular biology method and detected

In 4～6 leaf phases of seedling plant, get blade and carry out PCR and GUS and detect, according to the detected result in early stage, to 6-8 leaf during the phase, PCR is detected positive plant carry out Southern hybridization at the seedling plant strain growth, obtain to contain the transformed plant of target gene;

Step 7: the acquisition of the transfer-gen plant of destination gene expression

According to destination gene expression difference in period, Southern hybridization detection positive plant is carried out RT-PCR, Nouthern hybridization and Western hybridization detection in relevant period, simultaneously according to objective trait, screen the transfer-gen plant that obtains destination gene expression.

The present invention compares with the genetically modified result of study of reporting in the world of switchgrass, and in transformation receptor, Transformation Program, the transformation period, aspects such as the genotype scope of conversion have all obtained bigger raising, and effect is remarkable.Be in particular in:

(1) is easy to the transformation receptor that obtains

Callus is the acceptor that most of plant genetics transform.Because switchgrass tissue culture technique research relatively lags behind, making with the callus is that the widespread usage of agrobacterium-mediated transformation on switchgrass of transformation receptor has been subjected to considerable restraint.Seed is the blank of a young plant, has natural morphogenesis ability and hereditary transmission capacity.After the suction of exsiccant seed, the cells,primordial enzymic activity improves, and respiration is strengthened rapidly, and cell fission is vigorous, and nucleic acid, protein are synthetic again.And the synthetic and cell fission of DNA is the important factor that Agrobacterium successfully transforms plant.Therefore, utilize agrobacterium tumefaciens to act on the plant seed of sprouting, its T-DNA is shifted and be incorporated in the paotoblastic nucleus, thereby realize transforming.The present invention directly utilizes the seed of sprouting to be transformation receptor in view of the above, has set up the novel genetic conversion system of switchgrass, has overcome the existing Agrobacterium-mediated Transformation technology shortcoming strong to the conditions of tissue culture dependency.Simultaneously, mature seed can obtain all the year round, is not subjected to the restriction of the season of growth and greenhouse experiment, also provides a simple and easy to do approach for the switchgrass transgenic research.

(2) simplified Transformation Program

Compare with conventional agrobacterium-mediated transformation, method of the present invention is directly from the transformation receptor resistant buds of regenerating, do not need the differential period with resistant calli of inducing through embryo callus, simplified the program of genetic transformation, shortened the transformation period, more conventional agrobacterium-mediated transformation has reduced 100～120 days.Because the present invention does not need to increase extra expensive reagent, makes the corresponding reduction of experimental cost yet.

(3) widened the genotype scope of genetic transformation

Studies show that, also there is genotypic difference in switchgrass aspect artificial induction's callus, this makes the selection of switchgrass converting material be subjected to considerable restraint, cause present genetic transformation all to concentrate on indivedual genotype (mainly select at present genotype be Alamo), and these genotype to be not production practice required or use very limited on producing.The present invention directly utilizes the seed of sprouting to be transformation receptor, does not need inducing and differential period through callus.Facts have proved, can utilize to produce and go up existing all genotype, greatly widened the genotype scope of switchgrass genetic transformation, lay a good foundation for utilizing biotechnology that switchgrass is carried out genetic improvement.

Embodiment

The seed that the present invention utilizes switchgrass to sprout is transformation receptor, adopt agrobacterium-mediated transformation to transform and obtain switchgrass transgene plant, comprise the sterilization of seed, sprouting of seed, adopt agrobacterium-mediated transformation to transform, transformant is screened the root culture of resistance seedling, transplanting is carried out molecular biology method and is detected to the greenhouse, obtain the transfer-gen plant of destination gene expression according to objective trait.Specifically carry out as follows:

(1) sterilization of seed,

A. get the mature seed of switchgrass, the alcohol surface sterilization of employing 70% 1 minute;

B. adopt the Losantin (tween 80 of adding 0.1%) of 3% concentration, 500rpm/ minute, sterilized 2～2.5 hours;

C. use aseptic water washing 2～3 times, under 4 ℃ of conditions, secretly cultivated 12 hours;

D. adopt the Losantin of 3% concentration, 500rpm/ minute, two-stage sterilization 1～15 hour;

E. use aseptic water washing 2～3 times.

(2) seed sprouts

Seed is placed on on 4～5 layers of fully wetting aseptic filter paper of sterilized water, under 24 ℃ of conditions, secretly is cultured to seed sprout (showing money or valuables one carries unintentionally).

(3) Agrobacterium-mediated Transformation and common cultivation

A. the mono-clonal of picking Agrobacterium is in the LB of the 50mL that contains kantlex and Rifampin substratum, LB+kan (kantlex) 50mg/L+Rif (Rifampin) 25mg/L, 28 ℃, 200rpm/ minute～250rpm/ minute, cultivate 1～2 day to agrobacterium strains concentration be OD ₆₀₀=0.8～1.0; The LB substratum is: 10g tryptone, 5g yeast extract, 10g sodium-chlor, pH=7.0;

B. in substratum, add 50 μ l 100mM Syringylethanones, 28 ℃, 200-250rpm/ minute, cultivated 1～2 hour;

C. substratum is changed in the aseptic 50mL centrifuge tube, under the normal temperature, 3500rpm/ minute, centrifugal 12～15 minutes collection Agrobacteriums;

D. add 40～50mL liquid MBP substratum Agrobacterium that suspends again, regulating agrobacterium strains concentration is OD ₆₀₀=0.5.Liquid MBP substratum is: the mixing synthetics 4.43g/L of MS inorganic salt and VITAMIN, maltose 30g/L, 6-BA (6-benzylaminopurine) 3mg/L, pH=5.8;

The product that the mixing synthetics of above-mentioned MS inorganic salt and VITAMIN provides for reagent manufacturer.

E. laterally rive along the seed middle part with aseptic scalper, discard the half granule seed that does not contain radicle, add the good Agrobacterium bacterium liquid that suspends, add 50 μ l 100mM Syringylethanones simultaneously, after vacuumizing 60～90 minutes on the vaccum bench, under the normal temperature, 50rpm/ minute～60rpm/ minute cultivation 60～90 minutes;

F. discard whole Agrobacterium bacterium liquid, inhale with aseptic filter paper and remove the residual bacterium liquid of seed-coat, then seed is changed over to substratum MBP, 20～24 ℃, secretly cultivated 2～3 days.Wherein, the MBP substratum is: the mixing synthetics 4.43g/L of MS inorganic salt and VITAMIN, maltose 30g/L, 6-BA (6-benzylaminopurine) 3mg/L, agar powder 7.5g/L, pH=5.8; According to Agrobacterium growing state state, can suitably adjust common incubation time.Rule, the Agrobacterium growth is more, and incubation time is short altogether, otherwise, but then proper extension is total to incubation time.

(4) screening of resistant buds

A. seed is changed over to screening culture medium SMBP1, under 24 ℃ of conditions, 2～3 weeks of illumination cultivation; Wherein, screening culture medium SMBP1 is: the mixing synthetics 4.43g/L of MS inorganic salt and VITAMIN, maltose 30g/L, 6-BA (6-benzylaminopurine) 3mg/L, hpt (Totomycin) 100mg/L, Cefo (cephamycin) 250mg/L, agar powder 7.5g/L, pH=5.8;

B. seed is changed over to screening culture medium SMBP2, under 24 ℃ of conditions, 2～3 weeks of illumination cultivation; Wherein, screening culture medium SMBP2 is: the mixing synthetics 4.43g/L of MS inorganic salt and VITAMIN, maltose 30g/L, 6-BA (6-benzylaminopurine) 3mg/L, hpt (Totomycin) 75mg/L, Cefo (cephamycin) 250mg/L, agar powder 7.5g/L, pH=5.8;

C. seed is changed over to screening culture medium SMBP3, under 24 ℃ of conditions, 2～3 weeks of illumination cultivation; Wherein, screening culture medium SMBP3 is: the mixing synthetics 4.43g/L of MS inorganic salt and VITAMIN, maltose 30g/L, 6-BA (6-benzylaminopurine) 3mg/L, hpt (Totomycin) 50mg/L, Cefo (cephamycin) 250mg/L, agar powder 7.5g/L, pH=5.8; During this period, if find to have the seed or the young shoot of Agrobacterium pollution, in time will change new homotype screening culture medium screening over to other the normal seeds or the young shoot of ware.

(5) root culture of resistance seedling

, change among the root media SMO when 1～2cm is high at resistant buds (regrowth), 28 ℃, illumination condition is cultivated 3～4 weeks to seedling height down and is about more than the 5cm.Root media SMO is: mixing synthetics 2.22g/L, the sucrose 8g/L of MS inorganic salt and VITAMIN, agar powder 7.0g/L, pH=5.8.

(6) transplant to the greenhouse or the land for growing field crops is carried out molecular biology method and detected

Get blade in the seedling phase (4～6 leaf phase) and carry out PCR and GUS detection.According to the detected result in early stage, during the phase PCR is detected positive plant to the 6-8 leaf at the seedling plant strain growth and carry out Southern hybridization, obtain to contain the transformed plant of target gene.

(7) acquisition of the transfer-gen plant of destination gene expression

According to destination gene expression difference in period, Southern hybridization detection positive plant is carried out RT-PCR, Nouthern hybridization and Western hybridization detection in relevant period, simultaneously according to objective trait, screen the transfer-gen plant that obtains destination gene expression.

Overall operation flow process of the present invention is: the root culture → transplanting of the screening → resistance seedling of the sprouting of the sterilization → seed of seed → Agrobacterium-mediated Transformation and common cultivation → resistant buds to the greenhouse or the land for growing field crops carry out the acquisition of the transfer-gen plant of molecular biology method detection → destination gene expression.

It below is the specific embodiment that the contriver provides.

Embodiment

Delay gene P to contain leaf senile _SAG12The Agrobacterium-mediated Transformation GA9930 of-ipt, 3 switchgrass kinds of N.Switchgrass and Blackwell.Concrete conversion process is:

(1) sterilization of seed

A. get GA9930, the mature seed of N.Switchgrass and 3 switchgrass kinds of Blackwell, the alcohol surface sterilization of employing 70% 1 minute; Each kind is got 5000 seeds respectively, and experiment transforms 5 times altogether, transforms 1000 seeds at every turn;

B. adopt the Losantin (tween 80 of adding 0.1%) of 3% concentration, 500rpm/ minute, sterilized 2 hours;

C. use aseptic water washing 3 times, under 4 ℃ of conditions, secretly cultivated 12 hours;

D. adopt the Losantin two-stage sterilization 1.5 hours of 3% concentration;

E. use aseptic water washing 3 times.

(2) seed sprouts

Seed is placed on on 5 layers of fully wetting aseptic filter paper of sterilized water, under 24 ℃ of conditions, secretly is cultured to seed sprout (showing money or valuables one carries unintentionally); Wherein, the percentage of germination of GA9930 is about 85.5%, and N.Switchgrass is 90.1%, and Blackwell is 88.0%.

(3) Agrobacterium-mediated Transformation and common cultivation

A. picking contains P _SAG12Agrobacterium one mono-clonal of-ipt gene in the LB of the 50mL that contains kantlex substratum, LB+kan (kantlex) 50mg/L+Rif (Rifampin) 25mg/L, 28 ℃, 250rpm/ minute, cultivate 2 days to agrobacterium strains concentration be OD ₆₀₀=0.8～1.0; The LB substratum is: 10g tryptone, 5g yeast extract, 10g sodium-chlor, pH7.0;

B. in substratum, add 50 μ l 100mM Syringylethanones, 28 ℃, 250rpm/ minute, cultivated 2 hours;

C. substratum is changed in the aseptic 50mL centrifuge tube, under the normal temperature, 3500rpm/ minute, centrifugal 15 minutes collection Agrobacteriums;

D. add the 50mL liquid MBP substratum Agrobacterium that suspends again, regulating agrobacterium strains concentration is OD ₆₀₀=0.5.Liquid MBP substratum is: the mixing synthetics 4.43g/L of MS inorganic salt and VITAMIN, maltose 30g/L, 6-BA (6-benzylaminopurine) 3mg/L, pH=5.8;

E. laterally rive along the seed middle part with aseptic scalper, discard the half granule seed that does not contain radicle, add the good Agrobacterium bacterium liquid that suspends, add 50 μ l 100mM Syringylethanones simultaneously, after vacuumizing 60 minutes on the vaccum bench, under the normal temperature, cultivated 90 minutes in 60rpm/ minute;

F. discard whole Agrobacterium bacterium liquid, inhale with aseptic filter paper and remove the residual bacterium liquid of seed-coat, change the clump seed over to substratum MBP then, 24 ℃, secretly cultivated 2 days.Wherein, the MBP substratum is: the mixing synthetics 4.43g/L of MS inorganic salt and VITAMIN, maltose 30g/L, 6-BA (6-benzylaminopurine) 3mg/L, agar powder 7.5g/L, pH=5.8;

(4) screening of resistant buds

Change seed over to screening culture medium SMBP1 successively, among SMBP2 and the SMBP3, under 24 ℃ of conditions, successively 2～3 weeks of illumination cultivation, obtain resistant buds; Wherein:

A. seed is changed over to screening culture medium SMBP1, under 24 ℃ of conditions, 2 weeks of illumination cultivation; Wherein, screening culture medium SMBP1 is: the mixing synthetics 4.43g/L of MS inorganic salt and VITAMIN, maltose 30g/L, 6-BA (6-benzylaminopurine) 3mg/L, hpt (Totomycin) 100mg/L, Cefo (cephamycin) 250mg/L, agar powder 7.5g/L, pH=5.8;

B. seed is changed over to screening culture medium SMBP2, under 24 ℃ of conditions, 3 weeks of illumination cultivation; Wherein, screening culture medium SMBP2 is: the mixing synthetics 4.43g/L of MS inorganic salt and VITAMIN, maltose 30g/L, 6-BA (6-benzylaminopurine) 3mg/L, hpt (Totomycin) 75mg/L, Cefo (cephamycin) 250mg/L, agar powder 7.5g/L, pH=5.8;

C. seed is changed over to screening culture medium SMBP3, under 24 ℃ of conditions, 3 weeks of illumination cultivation; Wherein, screening culture medium SMBP3 is: the mixing synthetics 4.43g/L of MS inorganic salt and VITAMIN, maltose 30g/L, 6-BA (6-benzylaminopurine) 3mg/L, hpt (Totomycin) 50mg/L, Cefo (cephamycin) 250mg/L, agar powder 7.5g/L, pH=5.8; Wherein, GA9930, N.Switchgrass and Blackwell add up to 147 respectively, and the seed or the young shoot of 230 and 56 Agrobacterium pollutions are abandoned.

(5) root culture of resistance seedling

, change among the root media SMO when 1～2cm is high at resistant buds (regrowth), 28 ℃, illumination condition is cultivated 4 weeks to seedling plant height down and is about more than the 5cm.

5 conversions obtain 206 strain resistance seedlings altogether, and wherein GA9930 has 94 strains, N.Switchgrass 80 strains, Blackwell 32 strains.

(7) the molecular biology method detection is carried out in transplanting to the greenhouse

197 strains (wherein GA9930 has 89 strains, N.Switchgrass 80 strains, Blackwell 28 strains) of living that in the greenhouse, coexist of resistance seedling.Get blade in the seedling phase (5～6 leaf phase) and carry out PCR, obtain positive plant 163 strains altogether.During the phase PCR is detected positive plant to the 6-8 leaf at plant strain growth and carry out Southern hybridization, acquisition contains positive plant 148 strains of goal gene, and (wherein GA9930 has 70 strains, N.Switchgrass 57 strains, Blackwell 21 strains), transformation efficiency is about 1.12% (disregarding the seed of not sprouting with the Agrobacterium pollution).

(8) acquisition of the transfer-gen plant of destination gene expression

In the 7-8 leaf phase, Southern hybridization detection positive plant is carried out RT-PCR and Nouthern hybridization detection, wherein there is 121 strain RT-PCRs and Nouthern hybridization all to detect the purpose band in 148 strains, show that goal gene is expressed in these plant.

Claims (1)

1. a method that obtains switchgrass transgene plant is characterized in that, this method is a transformation receptor with the seed of sprouting, and adopts agrobacterium-mediated transformation to transform and obtains switchgrass transgene plant, specifically carries out as follows:

Step 1: the sterilization of seed

Get the mature seed of switchgrass, sterilize with the Losantin of 3% concentration, wherein add 0.1% tween 80 in the Losantin, the dark cultivation 12 hours used 3% Losantin two-stage sterilization again under 4 ℃ of conditions;

Step 2: the sprouting of seed

Seed on aseptic filter paper, under 24 ℃ of conditions, secretly is cultured to seed and sprouts;

Step 3: Agrobacterium-mediated Transformation and common cultivation

The mono-clonal of picking Agrobacterium under 28 ℃ of conditions, was cultivated 1～2 day with 200rpm/ minute～250rpm/ minute in the LB substratum that contains kantlex and Rifampin, to agrobacterium strains concentration be OD ₆₀₀=0.8～1.0, the LB substratum is: the 10g tryptone, and the 5g yeast extract, 10g sodium-chlor, pH are 7.0;

Adding Syringylethanone in the LB substratum continues to cultivate 1 hour～2 hours, change over to then in the aseptic 50mL centrifuge tube, under the normal temperature, with 3500rpm/ minute centrifugal 15 minutes collection Agrobacteriums, with the liquid MBP substratum Agrobacterium that suspends again, regulating agrobacterium strains concentration is OD ₆₀₀=0.5, wherein, liquid MBP substratum is: the mixing synthetics of MS inorganic salt and VITAMIN: 4.43g/L, maltose: 30g/L, 6-benzylaminopurine: 3mg/L, pH=5.8;

Laterally rive along the seed middle part, discard the half granule seed that does not contain radicle, add the good Agrobacterium bacterium liquid that suspends, add Syringylethanone simultaneously, after vacuumizing 60～90 minutes on the vaccum bench, under the normal temperature, with 50rpm/ minute～60rpm/ minute cultivation 60～90 minutes;

Discard whole Agrobacterium bacterium liquid, remove the residual bacterium liquid of seed-coat with the aseptic filter paper suction, then seed is changed over to substratum MBP, under 20 ℃～24 ℃ conditions, the dark cultivation 2～3 days, wherein, the MBP substratum is: mixing synthetics 4.43g/L, the maltose 30g/L of MS inorganic salt and VITAMIN, 6-benzylaminopurine 3mg/L, agar powder 7.5g/L, pH=5.8;

Step 4: the screening of resistant buds

Change seed over to screening culture medium SMBP1 successively, among SMBP2 and the SMBP3, under 24 ℃ of conditions, successively 2～3 weeks of illumination cultivation, obtain resistant buds; Wherein, screening culture medium SMBP1 is: mixing synthetics 4.43g/L, the maltose 30g/L of MS inorganic salt and VITAMIN, 6-benzylaminopurine 3mg/L, Totomycin 100mg/L, cephamycin 250mg/L, agar powder 7.5g/L, pH=5.8; Screening culture medium SMBP2 is: mixing synthetics 4.43g/L, the maltose 30g/L of MS inorganic salt and VITAMIN, 6-benzylaminopurine 3mg/L, Totomycin 75mg/L, cephamycin 250mg/L, agar powder 7.5g/L, pH=5.8; Screening culture medium SMBP3 is: mixing synthetics 4.43g/L, the maltose 30g/L of MS inorganic salt and VITAMIN, 6-benzylaminopurine 3mg/L, Totomycin 50mg/L, cephamycin 250mg/L, agar powder 7.5g/L, pH=5.8;

Step 5: the root culture of resistance seedling

When resistant buds growth 1cm～2cm is high, change among the root media SMO, 28 ℃, illumination condition was cultivated for 3～4 weeks down, to the seedling plant height be more than the 5cm, wherein, root media SMO is: mixing synthetics 2.22g/L, the sucrose 8g/L of MS inorganic salt and VITAMIN, agar powder 7.0g/L, pH=5.8;

Step 6: transplant to the greenhouse or the land for growing field crops is carried out molecular biology method and detected

In 4～6 leaf phases of seedling plant, get blade and carry out PCR and GUS and detect, according to the detected result in early stage, to 6-8 leaf during the phase, PCR is detected positive plant carry out Southern hybridization at the seedling plant strain growth, obtain to contain the transformed plant of target gene;

Step 7: the acquisition of the transfer-gen plant of destination gene expression

According to destination gene expression difference in period, Southern hybridization detection positive plant is carried out RT-PCR, Nouthern hybridization and Western hybridization detection in relevant period, simultaneously according to objective trait, screen the transfer-gen plant that obtains destination gene expression.