CN104745621B - The main method and its special culture media for planting transgenic wheat of China is obtained using Agrobacterium - Google Patents
The main method and its special culture media for planting transgenic wheat of China is obtained using Agrobacterium Download PDFInfo
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Abstract
The invention discloses a kind of the main method and its special culture media for planting transgenic wheat of China is obtained using Agrobacterium.The present invention provides the kit for being used to prepare genetically modified plants, including infect liquid, co-culture culture medium, calli induction media, differential medium and root media.Present invention also offers a kind of method of prepare transgenosis plant.The experiment proves that, wheat breed of the present invention for the main cultivation of China, optimize the culture medium of plant tissue culture, the present invention, which utilizes, infects liquid and one group of culture medium, and the immature embryo part of selection is as explant, the preparation of transgenic wheat has been carried out using these, improve the genetic transformation efficiency and frequency of the wheat breed of the main cultivation of China, conversion process enriches, working specification, the development of Wheat Transformation industrialization is substantially increased, especially promotes the development of the conversion industry of China's main breed wheat.
Description
Technical field
The present invention relates to bioengineering field, more particularly to a kind of obtain the main transgenic wheat of planting of China using Agrobacterium
Method and its special culture media.
Background technology
Wheat is one of cereal crops important in the world, with socio-economic development, grain security supply and human nutrition
Health is closely related.China is Wheat Production state the biggest in the world.Wheat is in Chinese production after corn and rice within 2010
Amount has reached 2,240,000 tons.At present, the effort for yield and quality of wheat being improved by conventional breeding means has been had difficulty in taking a step, especially
It is excavating per mu yield potentiality, is ensureing to produce stability etc. per year.Gus/bar channel genes wheats are obtained from Vasil in 1992 etc.
Since obtaining the first transgenic wheat, research of the genetic improvement breeding on wheat has significant progress.Country will be small within 2008
The genetic improvement of wheat adds great transgenosis special project, actively and prudently promotes the cultivation work for implementing transgenic wheat new varieties
Make.Genetic improvement of wheat breeding at present still mainly uses particle bombardment, but there are of high cost, copy number for the method being introduced directly into
More, target gene and riddled basins close linkage cause offspring to be difficult to separate and the shortcomings of gene silencing phenomenon are serious.
Another means of Agrobacterium-mediated Transformation method as genetic engineering breeding, achieve in the crops such as corn, rice and barley
Immense success.In contrast, wheat belongs to alloploid plant, and genome is huge, and repetitive sequence is more, and genotype effects are serious,
Agrobacterium-mediated Transformation is relatively difficult, this is also the slow-paced main cause of wheat cdna Engineering Breeding.
Although wheat Agrobacterium-mediated Transformation process has significant development in recent years, it is small to be also concentrated mainly on some American-European spring
On wheat variety, such as " Bobwhite ", " Fielder ", " Cadenza " and " Veer y5 ", it is difficult in the main cultivation wheat breed of China
On be applied.And it is domestic there is presently no reported in literature comprehensive system by all parts of the country side's main breed collect into
Row tissue cultures and genetic transformation test, it is more to be simply confined on some native breeds, and conversion process is single, operates not
Low etc. this of specification, transformation efficiency just greatly limit the development of Wheat Transformation industrialization.
The content of the invention
The object of the present invention is to provide a kind of kit for being used to prepare genetically modified plants.
Provided by the present invention for the kit of prepare transgenosis plant, including infect liquid, co-cultivation culture medium, callus and lure
Lead culture medium, differential medium and root media；
The liquid that infects is made of 1/10MS minimal mediums, ethyl sulfonic acid, 2,4- dichlorphenoxyacetic acids, maltose and water,
The ethyl sulfonic acid it is described infect liquid in final concentration of 0.1g/L, 2, the 4- dichlorphenoxyacetic acids infect liquid described
Final concentration of 5.0mg/L, the maltose it is described infect liquid in final concentration of 30g/L；
The co-cultivation culture medium is by 1/10MS minimal mediums, ethyl sulfonic acid, proline, 2,4- dichlorphenoxyacetic acids, wheat
Bud sugar, ascorbic acid, acetosyringone, coagulator and water composition；Wherein, the ethyl sulfonic acid is in the co-cultivation culture medium
Final concentration of 0.1g/L, final concentration of 0.69g/L of the proline in the co-cultivation culture medium, 2, the 4- dichloro-benzenes
Final concentration of 5.0mg/L of the fluoroacetic acid in the co-cultivation culture medium, the maltose is in the co-cultivation culture medium
Final concentration of 30g/L, final concentration of 10g/L of the coagulator in the co-cultivation culture medium, the ascorbic acid is in institute
State the final concentration of 100mg/L co-cultured in culture medium, final concentration of the acetosyringone in the co-cultivation culture medium
For 200 μM.
The calli induction media is by MS minimal mediums, ammonium nitrate, cupric sulfate pentahydrate, ethyl sulfonic acid, 2,4- dichloro-benzenes
Fluoroacetic acid, caseinhydrolysate, proline, maltose, coagulator, ascorbic acid, Ticarcillin/Clavulanate Acid and water composition, wherein, the nitric acid
Final concentration of 2.4g/L of the ammonium in the calli induction media, the cupric sulfate pentahydrate is in the calli induction media
Final concentration of 1.25mg/L, final concentration of 1.95g/L of the ethyl sulfonic acid in the calli induction media, described 2,4-
Final concentration of 2.0mg/L of the dichlorphenoxyacetic acid in the calli induction media, the caseinhydrolysate is in the callus
Final concentration of 1.0g/L in inducing culture, the proline final concentration are final concentration of in the calli induction media
0.69g/L, final concentration 40g/L of the maltose in the calli induction media, the coagulator are lured in the callus
Lead the final concentration 8g/L in culture medium, final concentration of 100mg/L of the ascorbic acid in the calli induction media, institute
State final concentration of 200mg/L of the Ticarcillin/Clavulanate Acid in the calli induction media；
The differential medium by MS minimal mediums, cupric sulfate pentahydrate, ethyl sulfonic acid, glutamine, sucrose, coagulator,
Thidiazuron, glufosinate-ammonium, Ticarcillin/Clavulanate Acid and water composition, wherein, the cupric sulfate pentahydrate is final concentration of in the differential medium
1.25mg/L, final concentration of 1.95g/L of the ethyl sulfonic acid in the differential medium, the glutamine is in the differentiation
Final concentration of 0.75g/L in culture medium, final concentration of 30g/L of the sucrose in the differential medium, it is described solidifying
Gu final concentration of 3g/L of the agent in the differential medium, end of the Thidiazuron in the differential medium
Concentration is 0.5mg/L, and final concentration of 5mg/L of the glufosinate-ammonium in the differential medium, the Ticarcillin/Clavulanate Acid is at described point
Change the final concentration of 200mg/L in culture medium；
The root media is by 1/2 MS minimal mediums, ethyl sulfonic acid, α-naphthylacetic acid, sucrose, coagulator, glufosinate-ammonium
Formed with Ticarcillin/Clavulanate Acid, wherein, final concentration of 0.5g/L of the ethyl sulfonic acid in the root media, the α-naphthylacetic acid exists
Final concentration of 0.5mg/L in the root media, final concentration of 30g/L of the sucrose in the root media,
Final concentration of 3g/L of the coagulator in the root media, end of the glufosinate-ammonium in the root media are dense
Spend for 5mg/L, final concentration of 200mg/L of the Ticarcillin/Clavulanate Acid in the root media.
In mentioned reagent box, the coagulator is agar, agarose or plant gel.
In mentioned reagent box, the coagulator co-cultured in culture medium is agarose, the calli induction media
In coagulator be agar；Coagulator in the differential medium and the root media is plant gel.
In mentioned reagent box, the pH value that liquid and each culture medium are infected described in the kit is 5.8.
It is a further object to provide a kind of method of prepare transgenosis plant.
Method provided by the invention, includes the following steps:
1) prepare explant after pretreatment and infect and use bacterium solution：
The method of explant includes the following steps after the preparation pretreatment：The explant of plant is placed in above-mentioned reagent
Infect in liquid, centrifuge, abandoning supernatant, explant after being pre-processed described in box；
The preparation is infected to be included the following steps with the method for bacterium solution：First the recombinational agrobacterium containing foreign gene is suspended
Resuspended bacterium solution is obtained in liquid in described infect, then by added in the resuspended bacterium solution final concentration of 200 μM acetosyringone and
Final concentration of 0.1%（Mass percentage, is the percentage of quality and volume）Poloxamer（Pluronic F68, general stream
Buddhist nun gram F68）, obtain infecting and use bacterium solution；
2）Explant after the pretreatment and described infect are mixed with bacterium solution, stood, obtains infecting rear explant；
3）By it is described infect rear explant it is described co-cultivation culture medium in co-cultured, obtain co-culture embryo；
4）The co-cultivation embryo is subjected to Fiber differentiation in the calli induction media, callus group after being induced
Knit；
5）Callus after the induction is subjected to differentiation culture in the differential medium, obtains growing the callus of green seedling
Tissue；
6）The callus for growing green seedling is subjected to culture of rootage on the root media, that is, obtains transgenosis
Plant.
In the above method, the explant is prepared as follows：The embryo of the seed of the plant is cut off into plumule, is protected
Embryo remainder is stayed, obtains explant.
In the above method, step 1）In, the centrifugation centrifuges 25-35min for 18000-22000G；The centrifugation is specially
20,000G centrifuges 30min；
The concentration of bacterium is OD600=1.0 ± 0.5 in the resuspended bacterium solution；
Step 2）In, described stand as 23-28 DEG C of standing 0.8-1.5h, the standing is specially 25 DEG C of standing 1h；
Step 3）In, the condition of the co-cultivation is 22-25 DEG C of light culture 2.5-3.5 days, and the condition of the co-cultivation has
Body is 23 DEG C of light cultures 3 days；
Step 4）In, the condition of the Fiber differentiation is 22-25 DEG C of light culture 2.5-3.5 weeks, the bar of the Fiber differentiation
Part is 24 DEG C of light cultures 3 weeks；
Step 5）In, the condition of the differentiation culture is 145-155 μm of 22-25 DEG C, intensity of illumination ol/m2/ s, photoperiod
16/8h is cultivated 3.5-4.5 weeks, and the condition of the differentiation culture is specially 150 μm of 24 DEG C, intensity of illumination ol/m2/ s, photoperiod
16/8h is cultivated 4 weeks；
Step 6）In, the condition of the culture of rootage is 145-155 μm of 22-25 DEG C, intensity of illumination ol/m2/ s, photoperiod
16/8h is cultivated 3.5-4.5 weeks, and the condition of the culture of rootage is specially 150 μm of 24 DEG C, intensity of illumination ol/m2/ s, photoperiod
16/8h is cultivated 4 weeks.
In the above method, step 3）In, described infect is equipped with filter paper interval between rear explant and the co-cultivation culture medium；
Step 4）In, the co-cultivation embryo is placed on the calli induction media by scultellum in a manner of upward to be lured
Lead culture；
Step 5）In, the differentiation culture is once carried out according to subculture every two weeks, the culture medium and training that the subculture uses
The condition of supporting is identical with the differentiation culture.
In the above method, the recombinational agrobacterium is that plasmid corotation is entered to the restructuring agriculture bar obtained in agrobacterium tumefaciens AGL1
Bacterium.Wherein plasmid is pAL154/156, and wherein plasmid pAL154 is transfection helper plasmid, and the physical map of plasmid pAL156 is as schemed
Shown in 1.
In the above method, the wheat seed is the wheat flowering seed of 14 days；
The wheat is specially Chinese main breed, China's main breed be especially specially Henan wheat 949, Gansu Province spring 23,
Shandong wheat 5, Huaihe River wheat 19 or section's agriculture 199.
Above-mentioned MS minimal mediums are existing conventional culture medium, its component is NH4NO3、KNO3、KH2PO4、MgSO4·
7H2O、CaCl2、FeSO4·7H2O、Na2EDTA、MnSO4·4H2O、ZnSO4·4H2O、H3BO3、KI、Na2MoO4.2H2O、
CuSO4.5H2O、CoCl2·6H2O, glycine, thiamine hydrochloride, pyridoxine hydrochloride, nicotinic acid and inositol, above-mentioned each component it is dense
Degree is also existing conventional concentration.
Above-mentioned 1/10MS minimal mediums are that each component is 1/10 in MS minimal mediums.
The experiment proves that the present invention is directed to the wheat breed of the main cultivation of China, the culture medium of plant tissue culture is optimized,
The present invention selects immature embryo part as explant using infecting liquid and one group of culture medium, has been carried out turn using these
The preparation of DNA triticum, compared with wheat breed Bobwhite, improves the genetic transformation efficiency of the wheat breed of the main cultivation of China
And frequency, conversion process enriches, working specification, substantially increases the development of Wheat Transformation industrialization, especially promotes China
The development of the conversion industry of main breed wheat.
Brief description of the drawings
Fig. 1 is the physical map of pAL156
Fig. 2 is the Material growth situation during transformed wheat rataria
Fig. 3 is the Southern hybridization check results of wheat transgenic plant
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples, is commercially available unless otherwise specified.
Wheat breed Henan agriculture 949：Record in the following literature：Liu Zhangqing, the gorgeous new variety of wheat of Cui Li --- Henan agriculture 949.
Agricultural science and technology communicates, and the 11 phase public can be obtained from Inst. of Genetics and Development Biology, CAS's Developmental Biology research within 2008.
Wheat breed Gansu Province spring 23：Record in the following literature：What sweet osmanthus, 23 selection and breeding of Yang Wen hero New Spring Wheat Variety Longchuns
Report agriculture of gansu science and technology, the 02 phase public can be obtained from Inst. of Genetics and Development Biology, CAS's Developmental Biology research within 2005.
Wheat breed Shandong wheat 5：Record in the following literature：Shandong Agricultural University's wheat breeding research department multi-resisting high-yields
No. 5, Shandong agricultural sciences of new variety of wheat --- Shandong wheat, the 03 phase public can give birth to from Chinese Academy of Sciences's heredity with development within 1985
Wu Xue research institutes obtain.
Wheat breed Huaihe River wheat 19：Record in the following literature：The feature and high-yield culturing skill of Fang Yuanlin Huaihe River wheats 19
Art modern agriculture science and technology, the 12 phase public can be obtained from Inst. of Genetics and Development Biology, CAS's Developmental Biology research within 2011.
Wheat breed section agriculture 199：Record in the following literature：Zhao Hui, Zhang Wei, Wang Jing, Ji Jun, Wang Zhiguo, Li Jun bright winters
" 199 " stable high yield signature analysis Chinese Ecological Agriculture journal 05 phases of the .2011 public of section's agriculture can be from China for new variety of wheat
Academy of sciences's heredity is obtained with Developmental Biology research.
Wheat breed Bobwhite：Record in the following literature：Cheng M,Fry JE,Pang SZ,Zhou HP,
Hironaka CM,Duncan DR,Conner TW,and Wa YC.Genetic Transformation of Wheat
Mediated by Agrobacterium tumefaciens.Plant Physiol.1997115:971-980. the public can be from
Inst. of Genetics and Development Biology, CAS obtains.
pAL154/156：Record in the following literature：Wu H,Sparks C,Amoah B,Jones HD.Factors
influencing successful Agrobacterium-mediated genetic transformation of
wheat.Plant Cell Rep.200321:The 659-668. public can be from Developmental Biology research institute of Inst. of Genetics and Development Biology, CAS
Obtain.
Agrobacterium tumefaciens strains A GL1：Record in the following literature：Lazo GR,Stein PA,Ludwig RA.A DNA
transformation-competent Arabidopsis genomic library in Agrobacterium.Bio-
Technology.19919:The 963-967. public can be obtained from Inst. of Genetics and Development Biology, CAS's Developmental Biology research.
Component in following embodiments in MS minimal mediums is as shown in table 1：
Table 1 is the component in MS culture mediums
| A great number of elements | Final concentration in culture medium（g·L-1） |
| NH4NO3 | 1.65 |
| KNO3 | 1.9 |
| KH2PO4 | 0.17 |
| MgSO4.7H2O | 0.37 |
| CaCl2 | 0.44 |
| Trace element | Concentration in culture medium（mg·L-1） |
| FeSO4.7H2O | 27.8 |
| Na2EDTA | 37.3 |
| MnSO4.4H2O | 22.3 |
| ZnSO4.4H2O | 8.6 |
| H3BO3 | 6.2 |
| KI | 0.83 |
| Na2MoO4.2H2O | 0.25 |
| CuSO4.5H2O | 0.025 |
| CoCl2.6H2O | 0.025 |
| Organic principle | Concentration in culture medium（mg·L-1） |
| Glycine | 2.0 |
| Thiamine hydrochloride | 0.4 |
| Pyridoxine hydrochloride | 0.5 |
| Nicotinic acid | 0.5 |
| Inositol | 100 |
The detection of GUS transient expressions situation is as follows in following embodiments：Material to be detected is taken to be immersed in X-Gluc buffer solutions,
37 DEG C of overnight incubations, detect the transient expression situation specific method such as document Jefferson RA.1987.Assaying of GUS
chimeric genes in plants:the GUS gene fusion system.Plant MolBiol Rep5:387-
Described in 405.
Quantitative result involved in following embodiments, is to test in triplicate, results are averaged.
Embodiment 1, utilize Agrobacterium-mediated Transformation method acquisition transgenic wheat
It is prepared by the kit for the first, being used to prepare genetically modified plants
Being used to prepare the kit of genetically modified plants includes infecting liquid, co-cultures culture medium, calli induction media, differentiation
Culture medium and root media；
Component and the preparation method for infecting liquid and each culture medium are as follows：
1）Infect liquid
Infect liquid to be made of 1/10MS minimal mediums, ethyl sulfonic acid, 2,4- dichlorphenoxyacetic acids, maltose and water, second sulphur
Final concentration of 0.1g/L of the acid in liquid is infected, final concentration of 5.0mg/L, malt of 2, the 4- dichlorphenoxyacetic acids in liquid is infected
Final concentration of 30g/L of the sugar in liquid is infected；
The above-mentioned liquid that infects is prepared as follows：The second of final concentration of 0.1g/L is added in 1/10MS minimal mediums
Sulfonic acid, the 2,4-D of final concentration of 5.0mg/L（2,4- dichlorphenoxyacetic acids）And the maltose of final concentration of 30g/L, mended with water
Sufficient volume, adjusts pH to 5.8, -4 DEG C is stored in after filtration sterilization.
2）Co-culture culture medium
Co-culture culture medium by 1/10MS minimal mediums, ethyl sulfonic acid, proline, 2,4- dichlorphenoxyacetic acids, maltose,
Ascorbic acid, acetosyringone, agarose and water composition；Wherein, final concentration of 0.1g/ of the ethyl sulfonic acid in culture medium is co-cultured
L, final concentration of 0.69g/L of the proline in culture medium is co-cultured, 2,4- dichlorphenoxyacetic acids are in culture medium is co-cultured
Final concentration of 5.0mg/L, final concentration of 30g/L of the maltose in culture medium is co-cultured, agarose is in culture medium is co-cultured
Final concentration of 10g/L, final concentration of 100mg/L and acetosyringone of the ascorbic acid in culture medium is co-cultured co-culturing
Final concentration of 200 μM in culture medium.
Above-mentioned co-cultivation culture medium is prepared as follows：First added in 1/10MS minimal mediums：It is final concentration of
The ethyl sulfonic acid of 0.1g/L, the proline of final concentration of 0.69g/L, 2, the 4-D of final concentration of 5.0mg/L and final concentration of 30g/
The maltose of L, adjusts pH to 5.8, adds the agarose of final concentration of 10g/L, 121 DEG C of autoclaving 20min, treat culture medium temperature
The ascorbic acid and 200 μM of acetosyringone of final concentration of 100mg/L is added when being down to 60 DEG C or so, solid culture is made
Base.
3）Calli induction media
Calli induction media is by MS minimal mediums, ammonium nitrate, cupric sulfate pentahydrate, ethyl sulfonic acid, 2,4- Dichlorophenoxy second
Acid, caseinhydrolysate, proline, maltose, agar, ascorbic acid, Ticarcillin/Clavulanate Acid and water composition, wherein, ammonium nitrate is lured in callus
Lead the final concentration of 2.4g/L in culture medium, final concentration of 1.25mg/L of the cupric sulfate pentahydrate in calli induction media, second
Final concentration of 1.95g/L of the sulfonic acid in calli induction media, 2,4- dichlorphenoxyacetic acids are in calli induction media
Final concentration of 2.0mg/L, final concentration of 1.0g/L of the caseinhydrolysate in calli induction media, proline final concentration is more
Hinder the final concentration of 0.69g/L in inducing culture, final concentration 40g/L of the maltose in calli induction media, agar exists
Final concentration 8g/L in calli induction media, final concentration of 100mg/L, Te Mei of the ascorbic acid in calli induction media
Final concentration of 200mg/L of the spit of fland in calli induction media；
Above-mentioned calli induction media is prepared as follows：Addition has following final concentration in MS minimal mediums
Each component：The ammonium nitrate of final concentration of 2.4g/L, the cupric sulfate pentahydrate of final concentration of 1.25mg/L, final concentration of 1.95g/L
Ethyl sulfonic acid, the 2,4-D of final concentration of 2.0mg/L, the caseinhydrolysate of final concentration of 1.0g/L, final concentration of 0.69g/L's
Proline, the maltose of final concentration of 40g/L, adjusts pH to 5.8, adds the agar of final concentration of 8g/L, 121 DEG C of autoclavings
20min, adds the ascorbic acid of final concentration of 100mg/L and the Te Mei of 200mg/L when culture medium temperature is down to 60 DEG C or so
Spit of fland, is made solid medium.
4）Differential medium
Differential medium is by MS minimal mediums, cupric sulfate pentahydrate, ethyl sulfonic acid, glutamine, sucrose, plant gel, thiophene
Benzene is grand, glufosinate-ammonium, Ticarcillin/Clavulanate Acid and water form, wherein, final concentration of 1.25mg/L of the cupric sulfate pentahydrate in differential medium, second
Final concentration of 1.95g/L of the sulfonic acid in differential medium, final concentration of 0.75g/L of the glutamine in differential medium,
Final concentration of 30g/L of the sucrose in differential medium, plant gel is final concentration of in differential medium
3g/L, final concentration of 0.5mg/L of the Thidiazuron in differential medium, final concentration of 5mg/ of the glufosinate-ammonium in differential medium
L, final concentration of 200mg/L of the Ticarcillin/Clavulanate Acid in differential medium；
Above-mentioned differential medium is prepared as follows：Add final concentration of 1.25mg/L's in MS minimal mediums
Cupric sulfate pentahydrate, the ethyl sulfonic acid of 1.95g/L, the glutamine of 0.75g/L, the sucrose of 30g/L, adjusts pH to 5.8, adds final concentration
For the plant gel of 3g/L, 121 DEG C of autoclaving 20min, are added final concentration of when culture medium temperature is down to 60 DEG C or so
The Ticarcillin/Clavulanate Acid of the Thidiazuron of 0.5mg/L, final concentration of 5mg/L glufosinate-ammoniums and final concentration of 200mg/L, are made solid medium.
5）Root media
Root media by 1/2 MS minimal mediums, ethyl sulfonic acid, α-naphthylacetic acid, sucrose, plant gel, glufosinate-ammonium and
Ticarcillin/Clavulanate Acid forms, wherein, final concentration of 0.5g/L of the ethyl sulfonic acid in root media, α-naphthylacetic acid is in root media
Final concentration of 0.5mg/L, final concentration of 30g/L of the sucrose in root media, end of the plant gel in root media
Concentration is 3g/L, final concentration of 5mg/L of the glufosinate-ammonium in root media, and Ticarcillin/Clavulanate Acid is final concentration of in root media
200mg/L。
Above-mentioned root media is prepared as follows：Added in 1/2 MS minimal mediums：It is final concentration of
The ethyl sulfonic acid of 0.5g/L, the α-naphthylacetic acid of final concentration of 0.5mg/L, the sucrose of final concentration of 30g/L, adjusts pH to 5.8, addition is eventually
Concentration is the plant gel of 3g/L, and 121 DEG C of autoclaving 20min, final concentration is added when culture medium temperature is down to 60 DEG C or so
For 5mg/L glufosinate-ammoniums and the Ticarcillin/Clavulanate Acid of 200mg/L, solid medium is made.
2nd, the acquisition of transgenic wheat
By the following method respectively with China main cultivation wheat breed Henan wheat 949, Gansu Province spring 23, Shandong wheat 5, Huaihe River wheat 19 and Ke Nong
199 and the part rataria of American-European kind Bobwhite are explant, using Agrobacterium by containing bar genes and gus genes
PAL154/156 cotransfections enter wheat cell, and Fig. 2 is the Material growth situation during transformed wheat rataria, wherein, Tu2AWei
It is placed in the wheat immature embryo co-cultured on base；Fig. 2 B are GUS transient expression situations after co-culturing three days；Fig. 2 C were rataria through three weeks
The callus formed after induction；The callus that Fig. 2 D are three weeks carries out GUS dyeing；Lifes of Fig. 2 E through differentiation culture after two weeks
Long situation；Fig. 2 F are the resistant plant through culture of rootage after two weeks；Fig. 2 G are the resistant plant after culture of rootage four weeks；Fig. 2 H are
The transfer-gen plant of greenhouse-grown after two months, can be normal solid.Fig. 2 I and 2J be respectively T0 for transfer-gen plant tender leaf,
Young pod GUS staining conditions.
Specific method is as follows：
1st, prepare explant after pretreatment and infect and use bacterium solution
1）The preparation of explant after pretreatment
A, the preparation of explant
Each wheat children tassel of the few flowering 14d of robust growth, pest and disease damage, strips young fringe kind in clip crop field or growth room
Son, with 75% alcohol surface sterilization 1min, aseptic water washing 1 time instills a drop Tween20 liquid, then with effective chlorine density 2%
Liquor natrii hypochloritis in jog 20min on 28 DEG C of shaking tables, aseptic water washing 6 times.It will be planted with scalpel in superclean bench
The embryo of son takes out, and cuts off plumule, and it is immature embryo to retain embryo remainder, is used to test in next step as explant, should
The size of immature embryo is 1.2-1.8mm, translucent.
B, explant after pre-processing
The immature embryo that will be stripped（About 200 pieces/pipe）It is placed in and fills in the 2.0ml centrifuge tubes that 1.0ml infects liquid, 20,
000G centrifuges 30min, and abandoning supernatant, retains explant after pretreatment.
2）Infect the preparation with bacterium solution
A, the acquisition of recombinational agrobacterium
By in pAL154/156 cotransfection agrobacterium tumefaciens AGL1, in 100mg/l kanamycins and 200mg/l carboxylic benzyl moulds
That grown in plain resistant panel is recombinational agrobacterium AGL1（pAL154/156）.
Picking recombinational agrobacterium AGL1（pAL154/156）Monoclonal bacterium colony, be inoculated in 10ml contain 100mg/l cards that
In the LB fluid nutrient mediums of mycin and 200mg/l carbenicillins, suspend in 28 DEG C of 200rpm and cultivate 24h, it is to be grown to OD600
Mix, and dispense into 1.5ml centrifuge tubes with 30% isometric glycerite when=1.0 or so（About 400-600 μ l are often managed）,
In -80 DEG C of freezen protectives.
B, the acquisition with bacterium solution is infected
a）Activation
The recombinational agrobacterium AGL1 that above-mentioned A is preserved（pAL154/156）With the LB fluid nutrient mediums of 5ml corresponding antibiotics
Mixing, 28 DEG C of 200rpm, which suspend, cultivates 9h progress thalline activation, draws 500 μ l activation bacterium solution and is uniformly coated on containing corresponding antibiosis
On the LB solid mediums of element, cultivated in the way of 28 DEG C of 2d+23 DEG C of 1d, the recombinational agrobacterium AGL1 activated
（pAL154/156）.
b）Infect the acquisition with bacterium solution
By the recombinational agrobacterium AGL1 of activation（pAL154/156）It was resuspended in the conversion same day to infecting in liquid, makes the concentration of bacterium
OD600=1.0 ± 0.5 is maintained at, obtains resuspended bacterium solution, then final concentration of 200 μM of acetosyringone is added into resuspended bacterium solution
With 0.1%（The percentage of quality and volume）Pluronic F68（Pluronic F68）, obtain infecting and use bacterium solution.
2nd, infect
To above-mentioned 1 processing after reservation pretreatment after explant centrifuge tube in add 1.0ml above-mentioned 1 obtain infect use
Bacterium solution, be slightly vortexed 30s, and 1h is stood at 25 DEG C, absorbs upper strata bacterium solution, obtains infecting rear explant.
3rd, co-culture
By above-mentioned 2 prepare infect rear explant after being dried on three layers of Whatman NO.1 qualitative filter papers, then be placed on
Co-culture on culture medium（The culture medium is directly provided with a Whatman NO.1 qualitative filter papers interval with infecting rear explant）, use
Tweezers are compacted（Fig. 2A）, co-cultured, obtain co-culturing embryo.
The condition of culture of co-cultivation is：Light culture 3d in 23 DEG C of biochemical cultivation cases.
Co-culture three days after GUS transient expressions situation as shown in Figure 2 B, it can be seen that gene is transferred to.
4th, the Fiber differentiation of callus
Above-mentioned 3 obtained co-cultivation embryos are moved on calli induction media, scultellum upward, 90 × 15mm per 25, ware,
Carry out Fiber differentiation, callus after being induced（Fig. 2 C）.
The plumule grown and fibrous root are pulled out in incubation in time and abandons the material of pollution.
The condition of Fiber differentiation is：In 24 DEG C of dark culturings 3 weeks.
Callus carries out GUS transient expressions situation as shown in Figure 2 D after induction, it can be seen that gene is transferred to.
5th, differentiation culture
Callus is moved on differential medium after the induction that above-mentioned 4 are obtained, and carries out differentiation culture, every two weeks subculture one
It is secondary, obtain growing the callus of green seedling（Fig. 2 E）.
Above-mentioned differentiation culture is 150 μm of 24 DEG C, intensity of illumination ol/m2Cultivated 4 weeks under the conditions of/s, photoperiod 16/8h；
The condition and culture medium that above-mentioned subculture is cultivated using differentiation.
And at first after two weeks, the callus of each clone is divided into four pieces, flock together culture, 90 ×
15mm is per 10 clones of ware.Material dead under pollution and screening pressure is abandoned in incubation in time.
6th, culture of rootage
The above-mentioned 5 obtained callus for growing green seedling are moved to culture of rootage is carried out on root media, taken root
Transgenic wheat, that is, obtain genetically modified plants.
The condition of above-mentioned culture of rootage is in 150 μm of 24 DEG C, intensity of illumination ol/m2Cultivated under the conditions of/s, photoperiod 16/8h
4 weeks.As shown in Figure 2 F, the resistant plant after culture of rootage four weeks is as shown in Figure 2 G for resistant plant through culture of rootage after two weeks.
The organization material of each clone is got together during culture of rootage, 90 × 15mm is cultivated for 3-9 per ware, is cut
Material dead under pollution and screening pressure is abandoned in incubation in time.
7th, strong sprout and the numerous growth of expansion
Treat the above-mentioned 6 obtained transgenic wheat aerial part length taken root to 10cm or so and induce more than 2 it is strong
During root system, transplant into the flowerpot of 12 × 12cm, in 500 μm of ol/m of intensity of illumination2/ s, photoperiod 16/8h, seedling stage diurnal temperature
It is T0 for transgenic wheat to strengthen seedling and propagating in 20/15 DEG C, booting and growth room that pustulation period diurnal temperature is 30/20 DEG C
（Fig. 2 H）.
Fig. 2 I and 2J are respectively that T0 is planted for transgenosis（Section's agriculture 199）The tender leaf of strain, young pod GUS staining conditions, it can be seen that
Gene is transferred to.
Above-mentioned experiment obtains the T0 of 6 kinds of wheat breeds for transgenic wheat：The T0 of Henan wheat 949 is for transgenic wheat, Gansu Province spring
23 T0 for transgenic wheat, Shandong wheat 5 T0 for transgenic wheat, Huaihe River wheat 19 T0 for transgenic wheat, the T0 of section's agriculture 199
For the T0 of transgenic wheat and Bobwhite for transgenic wheat.
3rd, the identification of transgenic wheat
1）PCR is detected
Use TIANGEN Quick-type plant genome DNA extraction system kits（Tiangeng biochemical technology (Beijing) limited public affairs
Department, catalogue numbering is L0814）Extract genes of the T0 for Transgenic plant of wheat of above-mentioned two obtained each wheat breeds
Group DNA, using respective genomic DNA as template, bar genes are target gene, with 5 '-GGATCTACCATGAGCCCAGA of special primer
- 3 ' and 5 '-TGCCTCCAGGGACTTCAG-3 ' carry out PCR amplifications, bar gene amplification fragments size is 356bp；Not turn base
The wheat plant genomic DNA of each kind of cause is negative control, using pAL154/156 as positive control, record PCR detections
The plant being positive.
As a result it is as follows：PCR product size is about 356bp equal for transgenic wheat PCR positive rates for the positive, 6 kind T0
More than 90%.
2）, Southern hybridization checks
From step 1）The T0 of the positive is accredited as Wheat Transformation plant, with CTAB methods（Stacey J,Isaac
P.1994.Isolation of DNA from plants.In:Isaac PG ed.Methods in molecular
biology——protocols for nucleic acid analysis by non-radioactive
probes.Totowa,NJ:Humana Press Inc.28:9-15）The genomic DNA of blade is extracted, is expanded with PCR in step 3
The bar genes of increasing（356bp）For probe, Southern hybridization checks are carried out, are as a result all positive.Wherein 16 plants of hybridization knot
Fruit is as shown in figure 3, wherein, swimming lane 1-16 is different T0 for transgenic wheat single plant（Section's agriculture 199）, NC is not carry out gene to turn
The wheat plant negative control of change, PC are pAL154/156 positive controls.
4th, transformation efficiency counts
Experiment in triplicate, counts the transformation efficiency of each wheat breed（Average value）.
Wherein：Transformation efficiency=it is above-mentioned two obtain T0 for transformed plant wheat immature embryo number/infect with wheat immature embryo number ×
100%。
The results are shown in Table 2：
Table 2 is transformation efficiency
As can be seen that compared with European varieties Bobwhite, the main transformation efficiency for planting wheat of China is all improved, and illustrates this
The culture medium and method of invention can improve the main transformation efficiency for planting wheat of China.
5th, Bar genescreens are tested
Prepare 0.5%（v/v）Basta（200mg/ml glufosinate-ammoniums）Solution, to 8 independences through step 3 test positive
Transfer-gen plant（Different wheat immature embryos is derived from, it is section's agriculture 199 that the kind of 1-8, which is,）T1 sprayed for plant shoots
Apply, count withered and yellow downright bad plant after a week（It is negative）With normal survival plant（It is positive）Quantity, segregation ratio is counted, as a result such as table
Shown in 3.
Table 3 is the segregation ratio of Bar genescreens experiment
Claims (12)
1. being used to prepare the kit of transgenic wheat, including infect liquid, co-culture culture medium, calli induction media, differentiation
Culture medium and root media；
The liquid that infects is made of 1/10 MS minimal mediums, ethyl sulfonic acid, 2,4- dichlorphenoxyacetic acids, maltose and water, described
Ethyl sulfonic acid it is described infect liquid in final concentration of 0.1g/L, 2, the 4- dichlorphenoxyacetic acids are dense at the end infected in liquid
Spend for 5.0mg/L, the maltose it is described infect liquid in final concentration of 30g/L；
The co-cultivation culture medium is by 1/10 MS minimal mediums, ethyl sulfonic acid, proline, 2,4- dichlorphenoxyacetic acids, malt
Sugar, ascorbic acid, acetosyringone, coagulator and water composition；Wherein, end of the ethyl sulfonic acid in the co-cultivation culture medium
Concentration is 0.1g/L, final concentration of 0.69g/L of the proline in the co-cultivation culture medium, 2, the 4- Dichlorophenoxies
Final concentration of 5.0mg/L of the acetic acid in the co-cultivation culture medium, end of the maltose in the co-cultivation culture medium
Concentration is 30g/L, and final concentration of 10g/L of the coagulator in the co-cultivation culture medium, the ascorbic acid is described
The final concentration of 100mg/L in culture medium is co-cultured, the acetosyringone is final concentration of in the co-cultivation culture medium
200μM；
The calli induction media is by MS minimal mediums, ammonium nitrate, cupric sulfate pentahydrate, ethyl sulfonic acid, 2,4- Dichlorophenoxy second
Acid, caseinhydrolysate, proline, maltose, coagulator, ascorbic acid, Ticarcillin/Clavulanate Acid and water composition, wherein, the ammonium nitrate exists
Final concentration of 2.4g/L in the calli induction media, end of the cupric sulfate pentahydrate in the calli induction media
Concentration is 1.25mg/L, the ethyl sulfonic acid final concentration of 1.95g/L in the calli induction media, 2, the 4- bis-
Final concentration of 2.0mg/L of the chlorophenoxyacetic acid in the calli induction media, the caseinhydrolysate are lured in the callus
The final concentration of 1.0g/L in culture medium is led, the proline final concentration is final concentration of in the calli induction media
0.69g/L, final concentration 40g/L of the maltose in the calli induction media, the coagulator are lured in the callus
Lead the final concentration 8g/L in culture medium, final concentration of 100mg/L of the ascorbic acid in the calli induction media, institute
State final concentration of 200mg/L of the Ticarcillin/Clavulanate Acid in the calli induction media；
The differential medium is by MS minimal mediums, cupric sulfate pentahydrate, ethyl sulfonic acid, glutamine, sucrose, coagulator, thiophene benzene
Grand, glufosinate-ammonium, Ticarcillin/Clavulanate Acid and water composition, wherein, the cupric sulfate pentahydrate is final concentration of in the differential medium
1.25mg/L, final concentration of 1.95g/L of the ethyl sulfonic acid in the differential medium, the glutamine is in the differentiation
Final concentration of 0.75g/L in culture medium, final concentration of 30g/L of the sucrose in the differential medium, it is described solidifying
Gu final concentration of 3g/L of the agent in the differential medium, end of the Thidiazuron in the differential medium
Concentration is 0.5mg/L, and final concentration of 5mg/L of the glufosinate-ammonium in the differential medium, the Ticarcillin/Clavulanate Acid is at described point
Change the final concentration of 200mg/L in culture medium；
The root media by 1/2 MS minimal mediums, ethyl sulfonic acid, α-naphthylacetic acid, sucrose, coagulator, glufosinate-ammonium and spy
Mei Ting is formed, wherein, final concentration of 0.5g/L of the ethyl sulfonic acid in the root media, the α-naphthylacetic acid is described
Final concentration of 0.5mg/L in root media, final concentration of 30g/L of the sucrose in the root media, it is described
Final concentration of 3g/L of the coagulator in the root media, the glufosinate-ammonium are final concentration of in the root media
5mg/L, final concentration of 200mg/L of the Ticarcillin/Clavulanate Acid in the root media.
2. kit according to claim 1, it is characterised in that：The coagulator is agar or agarose.
3. kit according to claim 1, it is characterised in that：The coagulator is plant gel.
4. kit according to claim 1, it is characterised in that：Coagulator in the co-cultivation culture medium is agar
Sugar, the coagulator in the calli induction media is agar；Solidification in the differential medium and the root media
Agent is plant gel.
5. according to any kits of claim 1-3, it is characterised in that：Liquid and each culture medium are infected in the kit
PH value be 5.8.
6. a kind of method of prepare transgenosis wheat, includes the following steps:
1) prepare explant after pretreatment and infect and use bacterium solution：
The method of explant includes the following steps after the preparation pretreatment：The explant of wheat is placed in claim 1-5
Infect in liquid, centrifuge, abandoning supernatant, explant after being pre-processed described in any kit；
The preparation is infected to be included the following steps with the method for bacterium solution：The recombinational agrobacterium containing foreign gene is first suspended in institute
State to infect and resuspended bacterium solution is obtained in liquid, then acetosyringone and end that final concentration of 200 μM are added in the resuspended bacterium solution is dense
Spend for 0.1%（Mass percentage）Poloxamer, obtain infecting and use bacterium solution；
2）Explant after the pretreatment and described infect are mixed with bacterium solution, stood, obtains infecting rear explant；
3）By it is described infect rear explant in claim 1-5 it is any it is described co-cultivation culture medium in co-cultured, obtain
Co-culture embryo；
4）The co-cultivation embryo is subjected to Fiber differentiation in any calli induction media in claim 1-5, is obtained
Callus after induction；
5）By callus after the induction, any differential medium carries out differentiation culture in claim 1-5, obtains
Grow the callus of green seedling；
6）The callus for growing green seedling is subjected to training of taking root in claim 1-5 on any root media
Support, that is, obtain genetically modified plants.
7. according to the method described in claim 6, it is characterized in that：The explant is prepared as follows：By the plant
The embryo excision plumule of the seed of thing, retains embryo remainder, obtains explant.
8. the method according to claim 6 or 7, it is characterised in that：
Step 1）In, the centrifugation centrifuges 25-35min for 18000-22000 G；
The concentration of bacterium is OD600=1.0 ± 0.5 in the resuspended bacterium solution；
Step 2）In, it is described to stand as 23-28 DEG C of standing 0.8-1.5h；
Step 3）In, the condition of the co-cultivation is 23 DEG C of light cultures 3 days；
Step 4）In, the condition of the Fiber differentiation is 22-25 DEG C of light culture 2.5-3.5 weeks, and the condition of the Fiber differentiation is
24 DEG C of light cultures 3 weeks；
Step 5）In, the condition of the differentiation culture is 145-155 μm of 22-25 DEG C, intensity of illumination ol/m2/ s, photoperiod 16/8h
Culture 3.5-4.5 weeks,；
Step 6）In, the condition of the culture of rootage is 145-155 μm of 22-25 DEG C, intensity of illumination ol/m2/ s, photoperiod 16/8h
Culture 3.5-4.5 weeks.
9. the method according to claim 6 or 7, it is characterised in that：Step 1）In, the centrifugation centrifuges for 20000G
30min；
The concentration of bacterium is OD600=1.0 ± 0.5 in the resuspended bacterium solution；
Step 2）In, it is described to stand as 25 DEG C of standing 1h；
Step 3）In, the condition of the co-cultivation is 23 DEG C of light cultures 3 days；
Step 4）In, the condition of the Fiber differentiation is 24 DEG C of light cultures 3 weeks；
Step 5）In, the condition of the differentiation culture is 150 μm of 24 DEG C, intensity of illumination ol/m2/ s, photoperiod 16/8h are cultivated 4 weeks；
Step 6）In, the condition of the culture of rootage is 150 μm of 24 DEG C, intensity of illumination ol/m2/ s, photoperiod 16/8h are cultivated 4 weeks.
10. the method according to claim 6 or 7, it is characterised in that：Step 3）In, the rear explant and described of infecting
Filter paper interval is equipped between co-cultivation culture medium；
Step 4）In, the co-cultivation embryo is placed on the calli induction media by scultellum in a manner of upward carries out induction training
Support；
Step 5）In, the differentiation culture is once carried out according to subculture every two weeks, the culture medium and culture bar that the subculture uses
Part is identical with the differentiation culture.
11. the method according to claim 6 or 7, it is characterised in that：The recombinational agrobacterium is that plasmid is transferred to root nodule agriculture
The recombinational agrobacterium obtained in bacillus AGL1.
12. the method according to claim 6 or 7, it is characterised in that：The wheat explant is chooses the small of flowering 14 days
Mai Yousui is prepared；
The wheat is Henan wheat 949, Gansu Province spring 23 or Shandong wheat 5.
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| 小麦不同外植体农杆菌转化体系研究;王艳丽;《中国优秀博硕士学位论文全文数据库（硕士） 农业科技辑》;20061015;第1.3.5.5节 * |
| 小麦组织培养再生相关基因克隆及分子特性分析;佘茂云;《中国博士学位论文全文数据库 农业科技辑》;20111015;第13页第1段 * |
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