CN108546711A - A kind of genetic transformation method for soybean - Google Patents
A kind of genetic transformation method for soybean Download PDFInfo
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Abstract
The invention belongs to Genetic Transformation in Higher Plants technical fields, specifically provide a kind of genetic transformation method for soybean, include the following steps：Transformation receptor is prepared, is prepared and is infected liquid, infect, co-culture, renewal cultivation, screening, extending culture and culture of rootage, the Genetic Transformation of Soybean plant with resistant gene of soybean is obtained.This method process is simple and convenient to operate, parameter and medium optimization, can obtain the Soybean transgenic plant of high genetic transformation rate within a short period of time.
Description
Technical field
The invention belongs to Genetic Transformation in Higher Plants technical fields, specifically provide a kind of genetic transformation method for soybean.
Background technology
Soybean is one of most widely used oil crops and cereal crops and human plant's property albumen and oil in the world
The important sources of fat, and soybean also plays an important role in the nitrogen cycle of biosphere.Genetically engineered soybean is that the mankind introduce earliest
One of crops of commercial growth, and maximum genetically modified plants of degree of commercialization highest, sown area so far,
It may be said that the genetic transfoumation of soybean is quite ripe.But remain that transformation efficiency is low, transformation time is long at present
The problems such as.
Invention content
In order to solve the above technical problem, the present invention provides a kind of genetic transformation method for soybean, by adjusting in the process
Culture medium and culture parameters, substantially reduce the time of Genetic Transformation of Soybean, and improve genetic transformation rate.
The invention is realized in this way providing a kind of genetic transformation method for soybean, include the following steps：
1) transformation receptor is prepared
The sterilizing of soya seeds：Soya seeds are taken, with 75% ethyl alcohol sterilizing 5min；Later use 15% hydrogen peroxide in
Shaking table 100rpm concussion processing 12min；Finally use sterile water wash 5 times；
Sprout culture：The sterile water 100ml for adding the 6-BA containing 2mg/L into the seed after sterilizing is placed in 4 DEG C of refrigerators dark
Culture 2 days；
2) it prepares and infects liquid
It takes 50ml to infect liquid bottom liquid, sequentially adds 100 100 100 50 μ of μ l, DTT50 μ l, ZT of μ l, AS of μ l, HCl of Cys
Mixed liquor is added in Agrobacterium by l, and Agrobacterium is made to suspend, and is made and is infected liquid；
3) it infects
The soybean for taking sprouting, is put into sterilizes culture dish, cuts away part hypocotyl, peels off kind of a skin, separates two panels cotyledon, clamps
There are the cotyledon of cotyledonary node, observation growing point position removes two panels true leaf, and point of a knife is drawn to growing point, and 1~2 knife is drawn,
Sterilizing triangular flask is taken, pours into infect liquid bottom liquid thereto, do not had bottom of bottle, the cotyledon pulled is put into sterilizing triangular flask；
The liquid that infects prepared by step 2) is added in the triangular flask for being placed with the soybean cotyledon for pulling wound, shaking table is placed
100rpm infects 3h；The back side is taken out later and is placed on aseptic filter paper upward blows 20min；
4) it co-cultures
Step 3) is blown to the cotyledon of surface no liquid, is connected to co-culture and be cultivated 4 days in base, the cotyledon back side downward, is trained altogether
It supports and places a sterilizing filter paper on base, the black print of the visible circle of soybean cotyledon node after co-cultivation, it is strong that growing point position grows bright yellow
Health bud, hypocotyl growth extend；
5) renewal cultivation
Cotyledon taking-up after co-cultivation is positioned in sterilizing ware, it is extra away from being cut at cotyledonary node 5mm in cotyledon hypocotyl
Hypocotyl, cotyledon oblique cutting is entered into renewal cultivation in recovery media later, renewal cultivation is after 5-7 days, until bud growth top is to going out
Culture medium；
6) it screens
A part of hypocotyl will be cut away at the standard compliant cotyledon ion leaf segment 5mm recovered, is inserted into screening training later
It supports and screens 15~21d in base, in screening process, the leaf on big bud gradually turns yellow, and constantly expands and have green at cotyledonary node
Color budlet constantly grows；
7) elongation culture
Conversion explant after screening is pulled out into yellow leaf, cotyledonary node bottom surface is cut away, then accesses elongation medium
Middle elongation culture, until the jaundice of big bud-leaf blacks, budlet jaundice；
8) culture of rootage
The seedling of elongation to 3-4cm in elongation medium are cut into taking-up, access root media is taken root.
Further, the gene that the Agrobacterium carries is bar resistant genes, epsps resistant genes or hyg resistant genes
In one kind；The soybean varieties are one kind in Jack, black agriculture 44, Williams82, DN50.
Further, it when soybean varieties are Jack or DN50, and the gene of Agrobacterium carrying is bar resistant genes, stretches
Long culture is divided into primary elongation and secondary extension, infects liquid bottom liquid, co-cultures base, recovery media, screening and culturing medium, once stretches
Long culture medium, secondary extension culture medium and root media are respectively J-sb-inf, J-sb-cocu, J-sb-reco, J-
Sbar2, J-E3bar0.5, J-E3+Tc and L5+Tc, one time elongation process is：Conversion explant after screening is pulled out into Huang
Leaf cuts away cotyledonary node bottom surface, 15~21d in an elongation medium J-E3bar0.5 is then accessed, until big bud is most of
Leaf jaundice blacks, budlet jaundice；Secondary extension process is：After primary elongation in access secondary extension culture medium J-E3+Tc into
30~45d of row elongation culture；
The culture medium specific formula used in each step see the table below：
Each code representative meaning in above-mentioned culture medium prescription table is：
Further, when soybean varieties be HN44 or Williams82, and Agrobacterium carry gene be bar resistant genes
When, elongation culture is divided into primary elongation and secondary extension, infect liquid bottom liquid, co-culture base, recovery media, screening and culturing medium,
Elongation medium, secondary extension culture medium, root media are respectively w-sb-inf, w-sb-cocu, w-sb-reco, w-
Sbar2, w-E5bar0.5, w-E5+Tc and L5+Tc, one time elongation process is：Conversion explant after screening is pulled out into Huang
Leaf cuts away cotyledonary node bottom surface, 15~21d in an elongation medium w-E5bar0.5 is then accessed, until big bud is most of
Leaf jaundice blacks, budlet jaundice；Secondary extension process is：After primary elongation in access secondary extension culture medium w-E5+Tc into
30~45d of row elongation culture；
The culture medium specific formula used in each step see the table below：
Each code representative meaning in above-mentioned culture medium prescription table is：
Further, when soybean varieties are Williams82, and the gene of Agrobacterium carrying is epsps resistant genes,
Elongation culture is divided into primary elongation and secondary extension, infects liquid bottom liquid, co-cultures base, is recovery media, screening and culturing medium, primary
Elongation medium, secondary extension culture medium, root media are respectively w-sb-inf, w-sb-cocu, w-sb-reco, w-
SE10, w-E5E5, w-E5+Tc and L5+Tc, one time elongation process is：Conversion explant after screening is pulled out into yellow leaf, is cut away
Then cotyledonary node bottom surface accesses 15~21d in an elongation medium w-E5E5, until big bud major part leaf jaundice hair
It is black, budlet jaundice；Secondary extension process is：After primary elongation elongation culture is carried out in access secondary extension culture medium w-E5+Tc
30~45d；The culture medium specific formula used in each step see the table below：
Each code representative meaning in above-mentioned culture medium prescription table is：
Further, when soybean varieties are Jack, and the gene of Agrobacterium carrying is epsps resistant genes, elongation training
Nutrient is primary elongation and secondary extension, infects liquid bottom liquid, co-cultures base, recovery media, screening and culturing medium, primary elongation training
It is respectively J-sb-inf, J-sb-cocu, J-sb-reco, J-SE10, J- to support base, secondary extension culture medium, root media
E3E5, J-E3+Tc and L5+Tc, one time elongation process is：Conversion explant after screening is pulled out into yellow leaf, cuts away cotyledonary node
Then bottom surface accesses 15~21d in an elongation medium J-E3E5, until the jaundice of big bud major part leaf blacks, budlet
Jaundice；Secondary extension process is：30~45d of elongation culture is carried out after primary elongation in access secondary extension culture medium J-E3+Tc；
The culture medium specific formula used in each step see the table below：
Each code representative meaning in above-mentioned culture medium prescription table is：
Further, when soybean varieties are Jack, and the gene of Agrobacterium carrying is hyg resistant genes, liquid bottom is infected
Liquid, co-cultivation base, recovery media, screening and culturing medium, elongation medium, root media are respectively w-sb-inf, w-sb-
Cocu, w-sb-reco, w-SH25, w-E5+Tc and L5+Tc, elongation process are：Conversion explant after screening is pulled out into Huang
Leaf cuts away cotyledonary node bottom surface, then accesses in elongation medium w-E5+Tc, and every 15~21d cycles subculture is primary；
The culture medium specific formula used in each step see the table below：
Each code representative meaning in above-mentioned culture medium prescription table is：
Further, what is prepared in the step 2) infects liquid OD600=0.8-1.0.
Further, the Agrobacterium kind is EHA105.
Compared with the prior art, the advantages of the present invention are as follows：Process is simple and convenient to operate, parameter and medium optimization, energy
Enough Soybean transgenic plants for obtaining high genetic transformation rate within a short period of time.
Description of the drawings
Fig. 1 is the soybean explant that embodiment 1 co-cultures the stage；
Fig. 2 is the soybean explant in 1 renewal cultivation stage of embodiment；
Fig. 3 is the positive bud that embodiment 1 induces；
Fig. 4 is that embodiment 1 is taken root the positive bud in stage.
Specific implementation mode
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiment, to the present invention
It is further elaborated.It should be appreciated that specific embodiment described herein is used only for explaining the present invention, it is not used to
Limit the present invention.
Embodiment 1, (soybean varieties DN50, it is bar resistant genes that Agrobacterium, which carries resistant gene)
Before the genetic transformation for carrying out soybean, first it was collecting bacteria, includes the following steps before one week：
1, single bacterium colony (2~3d) is drawn
Bacterium is taken out from -80 DEG C of refrigerators, picks and is a little spread out on YEP plates, 28 DEG C of back-off light cultures；
2, single bacterium colony is chosen
The bacterium of the 1mm individually to be grown fine with yellow pipette tips or sterilizing toothpick picking or so, pipette tips or toothpick, which are put into, later adds
In the glass tube for having 2mlYEP (added with antibiotic) liquid, 28 DEG C of shaking table 220r/min are put the tube into later, light culture, 16h~
24h；
3, it activates again
It draws 500 μ l bacterium solutions to be placed in 1.5mlEP pipes, meets at Molecular Detection department and be detected；100 μ l are drawn again to be placed in
In glass tube added with 2mlYEP (added with antibiotic) liquid, 28 DEG C of shaking table 220r, light culture, 8h, until bacterial concentration be 0.6~
0.9 just can be used；
4, bacterium is activated
Check that Molecular Detection report (selects bacterium standard：Electrophoretogram Marker is clear and band has mark, bacterium solution band clear
It can be seen that), 10~20 μ l of bacterium solution that the bacterial concentration that picking can use is 0.6~0.9 are put into 50ml YEP (added with antibiotic)
In, 28 DEG C of shaking table 220r, light culture, 16h~18h, until bacterial concentration is 0.6~0.9 just usable；
The specific genetic transformation process of soybean includes the following steps：
1) transformation receptor is prepared
The sterilizing of soya seeds：Soya seeds are taken, with 75% ethyl alcohol sterilizing 5min；Later use 15% hydrogen peroxide in
Shaking table 100rpm concussion processing 12min；Finally use sterile water wash 5 times；
Sprout culture：The sterile water 100ml for adding the 6-BA containing 2mg/L into the seed after sterilizing is placed in 4 DEG C of refrigerators dark
Culture 2 days；
2) it prepares and infects liquid
It takes 50ml to infect liquid bottom liquid, sequentially adds 100 100 100 50 μ of μ l, DTT50 μ l, ZT of μ l, AS of μ l, HCl of Cys
Mixed liquor is added in Agrobacterium (kind EHA105), Agrobacterium is made to suspend by l, is made and infects liquid, OD600=0.8-1.0；
3) it infects
The soybean for taking sprouting, is put into sterilizes culture dish, cuts away part hypocotyl, peels off kind of a skin, separates two panels cotyledon, clamps
There are the cotyledon of cotyledonary node, observation growing point position removes two panels true leaf, and point of a knife is drawn to growing point, and 1~2 knife is drawn,
Sterilizing triangular flask is taken, pours into infect liquid bottom liquid thereto, do not had bottom of bottle, the cotyledon pulled is put into sterilizing triangular flask；
The liquid that infects prepared by step 2) is added in the triangular flask for being placed with the soybean cotyledon for pulling wound, shaking table is placed
100rpm infects 3h；The back side is taken out later and is placed on aseptic filter paper upward blows 20min；
4) it co-cultures
Step 3) is blown to the cotyledon of surface no liquid, is connected to co-culture and be cultivated 4 days in base, the cotyledon back side downward, is trained altogether
It supports and places a sterilizing filter paper on base, the black print of the visible circle of soybean cotyledon node after co-cultivation, it is strong that growing point position grows bright yellow
Health bud, hypocotyl growth extends, with reference to figure 1；
5) renewal cultivation
Cotyledon taking-up after co-cultivation is positioned in sterilizing ware, it is extra away from being cut at cotyledonary node 5mm in cotyledon hypocotyl
Hypocotyl, cotyledon oblique cutting is entered into renewal cultivation in recovery media later, renewal cultivation is after 5-7 days, until bud growth top is to going out
Culture medium, with reference to figure 2；
6) it screens
A part of hypocotyl will be cut away at the standard compliant cotyledon ion leaf segment 5mm recovered, is inserted into screening training later
It supports and screens 15~21d in base, in screening process, the leaf on big bud gradually turns yellow, and constantly expands and have green at cotyledonary node
Color budlet constantly grows；
7) elongation culture
Elongation process：Conversion explant after screening is pulled out into yellow leaf, cotyledonary node bottom surface is cut away, then connects
Enter 15~21d in an elongation medium J-E3bar0.5, until the jaundice of big bud major part leaf blacks, budlet jaundice；
Secondary extension process：Carried out in access secondary extension culture medium J-E3+Tc after primary elongation elongation culture 30~
45d, with reference to figure 3；
8) culture of rootage
The seedling of elongation to 3-4cm in elongation medium are cut into taking-up, access root media is taken root, reference chart
4。
It infects liquid bottom liquid, co-culture base, recovery media, screening and culturing medium, an elongation medium, secondary extension culture
Base and root media be respectively J-sb-inf, J-sb-cocu, J-sb-reco, J-sbar2, J-E3bar0.5, J-E3+Tc and
L5+Tc；
The culture medium specific formula used in each step see the table below：
Each code representative meaning in above-mentioned culture medium prescription table is：
Embodiment 2,
The places different from embodiment 1 are, soybean varieties Jack.
Embodiment 3,
The places different from embodiment 1 are that soybean varieties HN44 infects liquid bottom liquid, co-cultures base, renewal cultivation
Base, screening and culturing medium, an elongation medium, secondary extension culture medium, root media are respectively w-sb-inf, w-sb-
Cocu, w-sb-reco, w-sbar2, w-E5bar0.5, w-E5+Tc and L5+Tc, one time elongation process is：After screening
Conversion explant pull out yellow leaf, cut away cotyledonary node bottom surface, then access an elongation medium w-E5bar0.5 in 15~
21d, until the jaundice of big bud major part leaf blacks, budlet jaundice；Secondary extension process is：Access secondary extension training after primary elongation
It supports and carries out 30~45d of elongation culture in base w-E5+Tc；
The culture medium specific formula used in each step see the table below：
Each code representative meaning in above-mentioned culture medium prescription table is：
Embodiment 4,
The places different from embodiment 3 are, soybean varieties Williams82.
Embodiment 5,
The places different from embodiment 1 are, soybean varieties Williams82, and the gene that Agrobacterium carries is
Epsps resistant genes infect liquid bottom liquid, co-culture base, recovery media, screening and culturing medium, an elongation medium, secondary stretch
Long culture medium, root media be respectively w-sb-inf, w-sb-cocu, w-sb-reco, w-SE10, w-E5E5, w-E5+Tc and
L5+Tc, one time elongation process is：Conversion explant after screening is pulled out into yellow leaf, cotyledonary node bottom surface is cut away, then connects
Enter 15~21d in an elongation medium w-E5E5, until the jaundice of big bud major part leaf blacks, budlet jaundice；Secondary extension mistake
Cheng Wei：30~45d of elongation culture is carried out after primary elongation in access secondary extension culture medium w-E5+Tc；
The culture medium specific formula used in each step see the table below：
Each code representative meaning in above-mentioned culture medium prescription table is：
Embodiment 6,
The places different from embodiment 5 are that soybean varieties are Heihe 45.
Embodiment 7,
The places different from embodiment 5 are that soybean varieties are black agriculture 66.
Embodiment 8,
The places different from embodiment 1 are, soybean varieties Jack, and the gene that Agrobacterium carries is epsps resistances
Gene, infect liquid bottom liquid, co-culture base, recovery media, screening and culturing medium, an elongation medium, secondary extension culture medium,
Root media is respectively J-sb-inf, J-sb-cocu, J-sb-reco, J-SE10, J-E3E5, J-E3+Tc and L5+Tc, and one
Secondary elongation process is：Conversion explant after screening is pulled out into yellow leaf, cuts away cotyledonary node bottom surface, then access is once stretched
15~21d in long culture medium J-E3E5, until the jaundice of big bud major part leaf blacks, budlet jaundice；Secondary extension process is：Once
It is accessed in secondary extension culture medium J-E3+Tc after elongation and carries out 30~45d of elongation culture；
The culture medium specific formula used in each step see the table below：
Each code representative meaning in above-mentioned culture medium prescription table is：
Embodiment 9,
The places different from embodiment 1 are, when soybean varieties are Jack, and the gene that Agrobacterium carries is hyg resistances
When gene, liquid bottom liquid being infected, co-culturing base, recovery media, screening and culturing medium, elongation medium, root media be respectively
W-sb-inf, w-sb-cocu, w-sb-reco, w-SH25, w-E5+Tc and L5+Tc, elongation process once, are only：It will screening
Later conversion explant pulls out yellow leaf, cuts away cotyledonary node bottom surface, then accesses in elongation medium w-E5+Tc, every 15
It is primary that~21d recycles subculture；
The culture medium specific formula used in each step see the table below：
Each code representative meaning in above-mentioned culture medium prescription table is：
Claims (9)
1. a kind of genetic transformation method for soybean, which is characterized in that include the following steps：
1) transformation receptor is prepared
The sterilizing of soya seeds：Soya seeds are taken, with 75% ethyl alcohol sterilizing 5min；Use 15% hydrogen peroxide in shaking table later
100rpm concussion processing 12min；Finally use sterile water wash 5 times；
Sprout culture：The sterile water 100ml for adding the 6-BA containing 2mg/L into the seed after sterilizing, is placed in light culture 2 in 4 DEG C of refrigerators
It；
2) it prepares and infects liquid
It takes 50ml to infect liquid bottom liquid, sequentially adds 100 100 100 50 50 μ l of μ l, ZT of μ l, DTT of μ l, AS of μ l, HCl of Cys, it will
Mixed liquor is added in Agrobacterium, and Agrobacterium is made to suspend, and is made and is infected liquid；
3) it infects
The soybean for taking sprouting, is put into sterilizes culture dish, cuts away part hypocotyl, peels off kind of a skin, separates two panels cotyledon, clamp there are
The cotyledon of cotyledonary node, observation growing point position remove two panels true leaf, and point of a knife is drawn to growing point, draws 1~2 knife, take and go out
Bacterium triangular flask pours into infect liquid bottom liquid thereto, do not had bottom of bottle, the cotyledon pulled is put into sterilizing triangular flask；
The liquid that infects prepared by step 2) is added in the triangular flask for being placed with the soybean cotyledon for pulling wound, shaking table 100rpm is placed and invades
Contaminate 3h；The back side is taken out later and is placed on aseptic filter paper upward blows 20min；
4) it co-cultures
Step 3) is blown to the cotyledon of surface no liquid, is connected to co-culture and be cultivated 4 days in base, the cotyledon back side downward, co-cultures base
One sterilizing filter paper of upper placement, the black print of the visible circle of soybean cotyledon node, growing point position grow bright yellow health bud after co-cultivation,
Hypocotyl growth extends；
5) renewal cultivation
By after co-cultivation cotyledon taking-up be positioned over sterilizing ware in, cotyledon hypocotyl away from cut at cotyledonary node 5mm it is extra under
Cotyledon oblique cutting is entered renewal cultivation in recovery media by plumular axis later, and renewal cultivation is after 5-7 days, until bud growth top is to going out culture
Base；
6) it screens
A part of hypocotyl will be cut away at the standard compliant cotyledon ion leaf segment 5mm recovered, is inserted into screening and culturing medium later
15~21d of middle screening, in screening process, the leaf on big bud gradually turns yellow, and is constantly expanded at cotyledonary node and have green small
Bud is constantly grown；
7) elongation culture
Conversion explant after screening is pulled out into yellow leaf, cuts away cotyledonary node bottom surface, then accesses in elongation medium and stretches
Long culture, until the jaundice of big bud-leaf blacks, budlet jaundice；
8) culture of rootage
The seedling of elongation to 3-4cm in elongation medium are cut into taking-up, access root media is taken root.
2. genetic transformation method for soybean as described in claim 1, which is characterized in that the gene that the Agrobacterium carries is bar
One kind in resistant gene, epsps resistant genes or hyg resistant genes；The soybean varieties be Jack, black agriculture 44,
One kind in Williams82, DN50.
3. genetic transformation method for soybean as claimed in claim 2, which is characterized in that when soybean varieties be Jack or DN50, and
When the gene that Agrobacterium carries is bar resistant genes, elongation culture is divided into primary elongation and secondary extension, infects liquid bottom liquid, altogether
Culture medium, recovery media, screening and culturing medium, an elongation medium, secondary extension culture medium and root media are respectively
J-sb-inf, J-sb-cocu, J-sb-reco, J-sbar2, J-E3bar0.5, J-E3+Tc and L5+Tc, an elongation process
For：Conversion explant after screening is pulled out into yellow leaf, cuts away cotyledonary node bottom surface, then accesses an elongation medium J-
15~21d in E3bar0.5, until the jaundice of big bud major part leaf blacks, budlet jaundice；Secondary extension process is：After primary elongation
30~45d of elongation culture is carried out in access secondary extension culture medium J-E3+Tc；
The culture medium specific formula used in each step see the table below：
Each code representative meaning in above-mentioned culture medium prescription table is：
4. genetic transformation method for soybean as claimed in claim 2, which is characterized in that when soybean varieties be HN44 or
Williams82, and when the gene that carries of Agrobacterium is bar resistant genes, elongation culture is divided into primary elongation and secondary extension,
Liquid bottom liquid is infected, base is co-cultured, recovery media, screening and culturing medium, an elongation medium, secondary extension culture medium, takes root
Culture medium is respectively w-sb-inf, w-sb-cocu, w-sb-reco, w-sbar2, w-E5bar0.5, w-E5+Tc and L5+Tc, and one
Secondary elongation process is：Conversion explant after screening is pulled out into yellow leaf, cuts away cotyledonary node bottom surface, then access is once stretched
15~21d in long culture medium w-E5bar0.5, until the jaundice of big bud major part leaf blacks, budlet jaundice；Secondary extension process is：
30~45d of elongation culture is carried out after primary elongation in access secondary extension culture medium w-E5+Tc；
The culture medium specific formula used in each step see the table below：
Each code representative meaning in above-mentioned culture medium prescription table is：
5. genetic transformation method for soybean as claimed in claim 2, which is characterized in that when soybean varieties be Williams82, and
When the gene that Agrobacterium carries is epsps resistant genes, elongation culture is divided into primary elongation and secondary extension, infect liquid bottom liquid,
Co-culturing base, recovery media, screening and culturing medium, an elongation medium, secondary extension culture medium, root media is respectively
W-sb-inf, w-sb-cocu, w-sb-reco, w-SE10, w-E5E5, w-E5+Tc and L5+Tc, one time elongation process is：It will sieve
Conversion explant after choosing pulls out yellow leaf, cuts away cotyledonary node bottom surface, then accesses in an elongation medium w-E5E5
15~21d, until the jaundice of big bud major part leaf blacks, budlet jaundice；Secondary extension process is：Secondary stretch is accessed after primary elongation
30~45d of elongation culture is carried out in long culture medium w-E5+Tc；
The culture medium specific formula used in each step see the table below：
Each code representative meaning in above-mentioned culture medium prescription table is：
6. genetic transformation method for soybean as claimed in claim 2, which is characterized in that when soybean varieties are Jack, and Agrobacterium
When the gene of carrying is epsps resistant genes, elongation culture is divided into primary elongation and secondary extension, infects liquid bottom liquid, co-cultures
Base, recovery media, screening and culturing medium, an elongation medium, secondary extension culture medium, root media are respectively J-sb-
Inf, J-sb-cocu, J-sb-reco, J-SE10, J-E3E5, J-E3+Tc and L5+Tc, one time elongation process is：It will screen
Conversion explant afterwards pulls out yellow leaf, cuts away cotyledonary node bottom surface, then access an elongation medium J-E3E5 in 15~
21d, until the jaundice of big bud major part leaf blacks, budlet jaundice；Secondary extension process is：Access secondary extension training after primary elongation
It supports and carries out 30~45d of elongation culture in base J-E3+Tc；
The culture medium specific formula used in each step see the table below：
Each code representative meaning in above-mentioned culture medium prescription table is：
7. genetic transformation method for soybean as claimed in claim 2, which is characterized in that when soybean varieties are Jack, and Agrobacterium
When the gene of carrying is hyg resistant genes, infects liquid bottom liquid, co-cultures base, recovery media, screening and culturing medium, elongation culture
Base, root media are respectively w-sb-inf, w-sb-cocu, w-sb-reco, w-SH25, w-E5+Tc and L5+Tc, are extended
Cheng Wei：Conversion explant after screening is pulled out into yellow leaf, cuts away cotyledonary node bottom surface, then accesses elongation medium w-E5
In+Tc, every 15~21d cycles subculture is primary；
The culture medium specific formula used in each step see the table below：
Each code representative meaning in above-mentioned culture medium prescription table is：
8. genetic transformation method for soybean as described in claim 1, which is characterized in that is prepared in the step 2) infects liquid
OD600=0.8-1.0.
9. genetic transformation method for soybean as described in claim 1, which is characterized in that the Agrobacterium kind is EHA105.
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| CN110073976A (en) * | 2019-05-07 | 2019-08-02 | 山西省农业科学院棉花研究所 | A kind of sterilization method of tissue culture soya seeds |
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| CN110073976A (en) * | 2019-05-07 | 2019-08-02 | 山西省农业科学院棉花研究所 | A kind of sterilization method of tissue culture soya seeds |
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