CN101173297B - High-efficiency genetic transformation method for soybean immaturity seed lobe regeneration system with auxiliary vacuum permeation - Google Patents
High-efficiency genetic transformation method for soybean immaturity seed lobe regeneration system with auxiliary vacuum permeation Download PDFInfo
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Abstract
The invention discloses an efficient genetic transformation method of vacuum permeation assistance soybean immature cotyledon regeneration system, relating to the technical field of plant genetic engineering, comprising the following steps: (a) the induction and proliferation of soybean immature cotyledon somatic embryo; (b) the vacuum permeation assistance introduction of foreign DNA; (c) the regeneration of the somatic embryo with foreign DNA into a plant. Adopting the vacuum permeation assistance agrobacterium mediating method, under a certain vacuum condition, after treat for a certain time, a plurality of micromechanical wounds are made on the soybean somatic embryo, which fully raises the intruding opportunity for agrobacterium to obviously raise the infection efficiency of agrobacterium and the conversion of the soybean compared with the common agrobacterium mediating method. Soybean immature cotyledon is used as an explant to transform genetically, which avoids the problem that chimerism easily, appears with the cotyledonary node as the explant.
Description
Technical field:
The present invention relates to plant genetic engineering field, disclose a kind of high-efficiency genetic transforming method of soybean immaturity seed lobe regeneration system with auxiliary vacuum permeation, thus the working method of improvement soybean heredity proterties.
Background technology:
The genetic transformation of soybean is one of difficult point of plant genetic engineering field always, though the report that much successfully obtains the soybean transgene plant is also arranged, soybean heredity transforms and never has breakthrough progress.Trace it to its cause, the most important is exactly that transformation efficiency does not significantly improve.
The method that is applied to the soybean heredity conversion at present mainly is divided into two classes: 1, direct method: mainly comprise particle bombardment, pollen tube passage method, electric shocking method, microinjection etc.; 2, support methods: mainly comprise agrobacterium tumefaciens-mediated transformation.What wherein obtain widespread use has only particle bombardment and agrobacterium tumefaciens-mediated transformation.Even these two kinds of methods also have certain limitation in application.The efficient conversion that how really to realize soybean remains the subject matter that this field faces.
The acceptor material that the Agrobacterium-mediated Transformation method adopts mostly is cotyledonary node greatly, and this system is by usefulness aseptic seedling cotyledonary node reported first success regeneration plants such as Cheng.There is its certain advantage in this system with respect to other indefinite buds and somatocyte system, but the plant mosaic of system's acquisition is many thus, and later stage screening operation amount is bigger.
The somatic embryos of soybean regeneration system rapidly obtains regeneration plant first and is reported in nineteen eighty-three by Christianson.Its explant that adopts is a plumular axis, and the explant that report after this adopts mostly is unmature subleaf and complete rataria greatly.But what really can be applied to transform be the system that Finer etc. is set up, unmature subleaf is placed on contains 40mg/L 2, induce the body embryo on the MS substratum of 4-D, embryonal connective tissue can or contain low-levelly 2 at inducing culture in the succeeding transfer culture process, breeds on the fluid suspension culture base of 4-D.Histologic analysis shows that newborn embryo is distributed in old embryo surface, be easy to transform, but and screen in the liquid medium within, become efficiently owing to the screening medium contact with the high area that can breed embryo to make to screen like this, thereby can overcome chimeric problem.After this many laboratories all adopt this system to obtain non-chimeric transfer-gen plant.Wherein Stewart has reported that in 1996 its Bt gene imports soybean and obtained the transfer-gen plant of pest-resistant evil.
Summary of the invention:
The present invention discloses a kind of high-efficiency genetic transforming method of soybean immaturity seed lobe regeneration system with auxiliary vacuum permeation, purpose be to overcome the existing low difficult point of soybean heredity transformation efficiency, adopt the soybean unmature subleaf as explant, a kind of auxiliary method that transforms foreign DNA of vacuum infiltration of utilizing is provided in Agrobacterium tumefaciens mediated method.
Concrete grammar of the present invention may further comprise the steps:
One, the inducing and breed of soybean unmature subleaf somatic embryo:
Employing 20～40mg/L 2,4-D inductive method is obtaining soybean unmature subleaf somatic embryo under dark culture condition, and makes its further propagation be the cotyledon type idiosome, the cotyledon type idiosome is changed in the sky culture dish carry out the drying cultivation under 25 ℃ of illumination conditions.
Two, the auxiliary foreign DNA that imports of vacuum infiltration:
Drying treatment 5-7 days idiosomes are immersed in the agrobacterium tumefaciens liquid (OD600=0.5), and rapid vacuumizing infected 5～15 minutes to-23Hg condition.Changing 1/2MS substratum+sucrose 30g/L+5.5g agar powder over to cultivated 3 days in (pH=6.5～7.0) altogether.
One, the acquisition of transfer-gen plant:
Explant after cultivating altogether is placed on the solid medium of the additional fungistat of 1/2MS substratum and cultivates, per 2 all subcultures once, when the resistance idiosome was sprouted plantlet, changing 1/2MS substratum+0.3～0.5mg/L NAA additional phase over to should carry out root culture on the antibiotic substratum; Practice transplantation of seedlings when growing the laggard row of the rhizome that physically well develops.
Positively effect of the present invention is: adopted vacuum infiltration to assist agrobacterium mediation method, under certain vacuum condition, after the certain hour processing, can on somatic embryos of soybean, make many small wounds, fully improved Agrobacterium invasion chance, obviously improved the efficiency of infection of Agrobacterium, compare with common agrobacterium-mediated transformation, significantly improved the transformation efficiency of soybean.In addition, the present invention adopts the soybean unmature subleaf to carry out genetic transformation as explant, is easy to generate chimeric problem when having avoided the employing cotyledonary node to transform as explant.
Description of drawings
Fig. 1 is the RT-PCR of embodiment 1 antisense PEP gene high-oil-content soybean part plant;
1: attached property control plasmid; 2: negative control; 3～8: genetically engineered soybean; M: molecular weight Marker
Fig. 2 is that the Southernblotting of embodiment 1 antisense PEP gene high-oil-content soybean part plant detects;
M: molecular weight Marker; +: plasmid;-: negative control (not genetically engineered soybean) P1-P3: antisense PEP gene soybean;
Fig. 3 is the pcr analysis that embodiment 2 soybean change two valency Bt gene plants;
1: molecular weight Maker; 2: plasmid DNA; 3:: negative control; 15: the non-transgenic material; 4-14,16-20: transgenic line
Fig. 4 detects (the HindIII enzyme is cut) for the Southern blotting of embodiment 2 transform insect-resistant gene soybean part plant;
M: molecular weight Maker; +: positive plasmid;-: negative control (not transfer-gen plant); 1-4: transfer-gen plant
Embodiment
By following examples the present invention is described for example further, and do not limit the present invention in any way, under the prerequisite that does not deviate from technical solution of the present invention, any change or change that those of ordinary skills that the present invention did are realized easily all will fall within the claim scope of the present invention.
Embodiment 1:
The importing of Jilin 35 somatic embryos generation and foreign gene
16～20 days soybean children pod that blooms is won in field planting Jilin 35, with aseptic water washing once, through 75% alcohol immersion 2 minutes, uses aseptic water washing again 3 times.Strip off kind skin is chosen 3～5mm cotyledon and is removed plumular axis, and the cotyledon adaxial and its surface upwards is inoculated in MS substratum+2, among the 4-D 20mg/L+ sucrose 3.0g/L+ plant gel 3.0g/L (pH=6.0), cultivates 14-30 days under 25 ℃ of dark conditions.
Choose the bright and green embryoid spherical in shape of color and luster, move to MS substratum+2, among the 4-D 5.0mg/L+ asparagine 1.0g/L+ sucrose 3.0g/L+ plant gel 3.0g/L (pH=5.8), 25 ℃ of illumination cultivation (16hr/8hr), per two all subcultures are once.
Somatic embryo is forwarded on MS substratum+gac 4.0g/L+ maltose 60.0g/L+ plant gel 3.0g/L (pH=6.4) to 25 ℃ of illumination cultivation (16hr/8hr).Per two all subcultures once.
When somatic embryo length arrives 2-3mm, it is changed on MS substratum+gac 4.0g/L+ maltose 60.0g/L+ plant gel 3.0g/L (pH=6.4), 25 ℃ of illumination cultivation (16hr/8hr), the inducing soybean somatic embryo is sprouted.Cultivated through 30 days, obtain the cotyledon type idiosome.Remove gac and continue to cultivate in substratum, after 4 weeks, cotyledon type idiosome color transition is cream-colored, it is changed in the sky culture dish carry out drying treatment under 25 ℃ of illumination conditions.
Drying treatment 5-7 days idiosomes are immersed the agrobacterium tumefaciens bacterium liquid (OD for preparing
600=0.5) in, rapid vacuumizing infected 10 minutes to-23Hg condition.Material is taken out, be placed on the filter paper bacterium liquid is blotted, change in 1/2MS substratum+sucrose 30g/L+5.5g agar powder (pH=7.0) and cultivated altogether 3 days.
Explant after cultivating altogether is placed on 1/2MS substratum+cephamycin 250mg/L+ Pyocianil 250mg/L+ kantlex 80 mg/L+ plant gel 3.0g/L (pH7.0) substratum to be cultivated, after 10 days, the resistance idiosome changes among the 1/2MS+NAA0.5mg/L+ cephamycin 250mg/L+ Pyocianil 250mg/L+ kantlex 100mg/L+ plant gel 3.0g/L (pH7.0) when sprouting plantlet and carries out root culture.The good regrowth of will taking root after 15 days is practiced transplantation of seedlings.
2 parts of the high-oil-content soybean materials of PEP gene have been obtained at present to change.See Fig. 1,2.
Embodiment 2:
The importing of Jilin 47 somatic embryos generation and foreign gene
16-20 days soybean children pod that blooms is won in field planting Jilin 47, with aseptic water washing once, through 75% alcohol immersion 2 minutes, uses aseptic water washing again 3 times.Strip off kind skin is chosen the 3-5mm cotyledon and is removed plumular axis, and the cotyledon adaxial and its surface upwards is inoculated in MS substratum+2, among the 4-D 40mg/L+ sucrose 3.0g/L+ plant gel 3.0g/L (pH=6.0), cultivates 14-30 days under 25 ℃ of dark conditions.
Choose the bright and green embryoid spherical in shape of color and luster, move to MS substratum+2, among the 4-D 5.0mg/L+ asparagine 1.0g/L+ sucrose 3.0g/L+ plant gel 3.0g/L (pH=5.8), 25 ℃ of illumination cultivation (16hr/8hr), per two all subcultures are once.
Somatic embryo is forwarded on MS substratum+gac 4.0g/L+ maltose 60.0g/L+ plant gel 3.0g/L (pH=6.4) to 25 ℃ of illumination cultivation (16hr/8hr).Per two all subcultures once.
When somatic embryo length arrives 2-3mm, it is changed on MS substratum+gac 4.0g/L+ maltose 60.0g/L+ plant gel 3.0g/L (pH=6.4), 25 ℃ of illumination cultivation (16hr/8hr), the inducing soybean somatic embryo is sprouted.Cultivated through 30 days, obtain the cotyledon type idiosome.Remove gac and continue to cultivate in substratum, after 4 weeks, cotyledon type idiosome color transition is cream-colored, it is changed in the sky culture dish carry out drying treatment under 25 ℃ of illumination conditions.
Drying treatment 5-7 days idiosomes are immersed the agrobacterium tumefaciens bacterium liquid (OD for preparing
600=0.5) in, rapid vacuumizing infected 20 minutes to-23Hg condition.Material is taken out, be placed on the filter paper bacterium liquid is blotted, change in 1/2MS substratum+sucrose 30g/L+5.5g agar powder (pH=7.0) and cultivated altogether 3 days.
Explant after cultivating altogether is placed on 1/2MS substratum+cephamycin 250mg/L+ Pyocianil 250mg/L+ kantlex 80mg/L+ plant gel 3.0g/L (pH7.0) substratum to be cultivated, after 10 days, the resistance idiosome is transferred among the 1/2MS+NAA0.5mg/L+ cephamycin 250mg/L+ Pyocianil 250mg/L+ kantlex 100mg/L+ plant gel 3.0g/L (pH7.0) when sprouting plantlet and carries out root culture.The good regrowth of will taking root after 15 days is practiced transplantation of seedlings.
2 parts of the soybean materials of trans Bt gene have been obtained at present.See Fig. 3,4.
Embodiment 3:
The importing of Jilin 20 somatic embryos generation and foreign gene
16-20 days soybean children pod that blooms is won in field planting Jilin 20, with aseptic water washing once, through 75% alcohol immersion 2 minutes, uses aseptic water washing again 3 times.Strip off kind skin is chosen the 3-5mm cotyledon and is removed plumular axis, and the cotyledon adaxial and its surface upwards is inoculated in MS substratum+2, among the 4-D 30mg/L+ sucrose 3.0g/L+ plant gel 3.0g/L (pH=6.0), cultivates 14-30 days under 25 ℃ of dark conditions.
Choose the bright and green embryoid spherical in shape of color and luster, move to MS substratum+2, among the 4-D 5.0mg/L+ asparagine 1.0g/L+ sucrose 3.0g/L+ plant gel 3.0g/L (pH=5.8), 25 ℃ of illumination cultivation (16hr/8hr), per two all subcultures are once.
Somatic embryo is forwarded on MS substratum+gac 4.0g/L+ maltose 60.0g/L+ plant gel 3.0g/L (pH=6.4) to 25 ℃ of illumination cultivation (16hr/8hr).Per two all subcultures once.
When somatic embryo length arrives 2-3mm, it is changed on MS substratum+gac 4.0g/L+ maltose 60.0g/L+ plant gel 3.0g/L (pH=6.4), 25 ℃ of illumination cultivation (16hr/8hr), the inducing soybean somatic embryo is sprouted.Cultivated through 30 days, obtain the cotyledon type idiosome.Remove gac and continue to cultivate in substratum, after 4 weeks, cotyledon type idiosome color transition is cream-colored, it is changed in the sky culture dish carry out drying treatment under 25 ℃ of illumination conditions.
Drying treatment 5-7 days idiosomes are immersed the agrobacterium tumefaciens bacterium liquid (OD for preparing
600=0.5) in, rapid vacuumizing infected 15 minutes to-23Hg condition.Material is taken out, be placed on the filter paper bacterium liquid is blotted, change in 1/2MS substratum+sucrose 30g/L+5.5g agar powder (pH=7.0) and cultivated altogether 3 days.
Explant after cultivating altogether is placed on 1/2MS substratum+cephamycin 250mg/L+ Pyocianil 250mg/L+ kantlex 80mg/L+ plant gel 3.0g/L (pH7.0) substratum to be cultivated, after 10 days, the resistance idiosome is transferred among the 1/2MS+NAA0.5mg/L+ cephamycin 250mg/L+ Pyocianil 250mg/L+ kantlex 100mg/L+ plant gel 3.0g/L (pH7.0) when sprouting plantlet and carries out root culture.The good regrowth of will taking root after 15 days is practiced transplantation of seedlings.
Obtained at present 1 part of the soybean material of trans Bt gene.
Can be suitable for multiple soybean genotype with method of the present invention, can transform various foreign genes, can obtain the transfer-gen plant of high conversion fast.
Claims (1)
1. the genetic transforming method of a soybean immaturity seed lobe regeneration system with auxiliary vacuum permeation comprises the steps:
(a) take away and spend back about 20 days soybean children pod, alcohol surface sterilization with 75%;
(b) cotyledon is removed plumular axis, adaxial and its surface upwards is inoculated in MS substratum+2,4-D 20mg/L+ sucrose 3.0g/L+ plant gel 3.0g/L, and pH is 6.0, dark condition was cultivated 14-30 days down;
(c) choose the bright and green embryoid spherical in shape of color and luster, move to the MS substratum and add 2,4-D5.0mg/L+ asparagine 1.0g/L+ sucrose 3.0g/L+ plant gel 3.0g/L, pH is 5.8,25 ℃ of illumination cultivation, per two all subcultures are once;
(d) somatic embryo is forwarded to MS substratum+gac 4.0g/L+ maltose 60.0g/L+ plant gel 3.0g/L, pH is 6.4,25 ℃ of illumination cultivation, and per two all subcultures once;
(e) the cotyledon type idiosome that grows is put into MS substratum+maltose 60.0g/L+ plant gel 3.0g/L, pH is 6.4,25 ℃ of illumination cultivation;
(f) after 4～6 weeks, treat that cotyledon type idiosome color transition carries out the drying cultivation for cream-colored it is changed in the aseptic empty culture dish under 25 ℃ of illumination conditions;
(g) drying treatment 5-7 days idiosome is immersed in the agrobacterium tumefaciens bacterium liquid, rapid vacuumizing infected 10 minutes to-23Hg condition; Change 1/2MS substratum+sucrose 30g/L+5.5g agar powder over to, pH is 7.0, cultivates altogether 3 days;
(h) explant that is total to after cultivating changes 1/2MS substratum+cephamycin 250mg/L+ Pyocianil 250mg/L+ kantlex 80mg/L+ plant gel 3.0g/L over to, pH is 7.0, cultivate, per 2 all subcultures once, when the resistance idiosome is sprouted plantlet, change 1/2MS+NAA0.5mg/L+ cephamycin 250mg/L+ Pyocianil 250mg/L+ kantlex 100mg/L+ plant gel 3.0g/L over to, pH is 7.0, carries out root culture; When growing the laggard capable acclimatization and transplants of the rhizome that physically well develops.
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| CN101880686B (en) * | 2010-07-12 | 2012-05-23 | 山东大学 | Vacuum-infiltration assisted exogenous gene transforming method of soybean germinating embryo |
| CN101974561A (en) * | 2010-11-23 | 2011-02-16 | 南京农业大学 | Genetic transformation method for soybean cotyledonary nodes by agrobacterium |
| CN102766650A (en) * | 2012-07-03 | 2012-11-07 | 吉林大学 | Agrobacterium rhizogenes-mediated and vacuum infiltration-assisted soybean genetic transformation method |
| CN103125391B (en) * | 2013-03-07 | 2015-06-17 | 河北农业大学 | Local one-year multi-generation breeding method of soybeans |
| CN104195170B (en) * | 2014-09-16 | 2016-04-13 | 云南省农业科学院生物技术与种质资源研究所 | A kind of paddy rice vacuum infiltration genetic transforming method |
| CN106480087B (en) * | 2016-12-30 | 2020-01-03 | 上海交通大学 | Soybean genetic transformation method for vacuum-assisted agrobacterium infection |
| CN107549016A (en) * | 2017-10-17 | 2018-01-09 | 沈阳善达生物科技有限公司 | The rooting method and rooting induction culture medium of a kind of soybean transgene tissue-cultured seedling |
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