CN101265480B - Conversion method for overcoming agrobacterium tumefaciens mediated wheat immature embryo browning and special culture medium for the same - Google Patents
Conversion method for overcoming agrobacterium tumefaciens mediated wheat immature embryo browning and special culture medium for the same Download PDFInfo
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Abstract
The invention discloses a wheat rataria conversion method mediated by agrobacterium tumefaciens and a special culture medium thereof. The wheat rataria conversion method mediated by agrobacterium tumefaciens provided by the method comprises the following steps: (1) the wheat rataria is inoculated in a culture medium for preincubation; the wheat rataria is cultivated in dark under the temperature between 24 DEG C and 26 DEG C for 4 days to 7 days; (2) the target agrobacterium tumefaciens are used to infect the wheat rataria which is cultivated in the step (1) to process co-culture, the target T-DNA in the agrobacterium tumefaciens is introduced to the wheat rataria. Foreign gene is introduced to the wheat by the conversion method mediated by the agrobacterium tumefaciens. After measurement, by using the culture medium and the conversion method provided by the method, embryonic callus inductivity and achievement rate of resistant plants are both enhanced.
Description
Technical field
The present invention relates to a kind of method for transformation and special-purpose its substratum thereof that overcomes agrobacterium tumefaciens mediated wheat immature embryo browning.
Background technology
The molecular biology research that is found to be of dna double spirane structure is laid a good foundation, and the foundation of DNA recombinant technology makes molecular biology research enter practice by theory, and plant genetic engineering research is arisen at the historic moment.On this basis, Zambryski etc. utilize agrobacterium-mediated transformation to obtain the first strain transgenic tobacco plant in the world, Horch etc. have set up agriculture bacillus mediated leaf disc transformation method, and dicotyledonous model plant such as potato, tomato, Arabidopis thaliana successively transgenosis is succeedd.After this, plant transgenic technology develops rapidly, PEG mediated method, the electricity successively set up except that agrobacterium-mediated transformation swash perforation method, virus-mediated method, particle bombardment, microinjection, pollen tube passage method and supersonic method etc., the species of transgenosis success constantly enlarge, plant for more than 50 that relate to 35 sections, totally 120 various plants.Through for many years the practice and the survival of the fittest, most method for transformation are progressively abandoned, have formed with agrobacterium-mediated transformation and particle bombardment prevailing two big plant transgene systems.Up to now, the transgenic plant of media agrobacterium co-cultivation acquisition account for about 85% of transgenic plant sum.Simultaneously, there is the foreign gene of the value utilized to import plant some, and progressively transgenic plant is applied in the agriculture production.Plant genetic engineering research has related to all respects of breed improvement, comprises pest-resistant, disease-resistant (virus, bacterium and fungal disease), antiweed, degeneration-resistant (cold, saline and alkaline, heavy metal etc.), quality-improving (carbohydrate, grease and protein etc.), developmental regulation and dietetic alimentation etc.When carrying out staple crops transgenic technology system and functional gene Study on Transformation, the industrialization area of crop new variety such as genetically engineered soybean, corn, cotton, rape on producing increases year by year.
In food crop, wheat belongs to the genetic transformation crop of difficulty the most, adds that transgenic research starts late, and the genetic engineering breeding process lags significantly behind other crop.(1991) such as Hessd etc. (1990), Mooney are successively studied the feasibility of agrobacterium-mediated transformation transformed wheat, but fail to obtain transfer-gen plant.Along with the appearance of particle gun, new selectable marker gene and the utilization of efficient promoter, wheat transgenic research begins to increase after 1991.Vasil in 1992 etc. utilize the particle gun mediated method that the bar gene has been imported wheat, have obtained the first routine transgenic wheat plant in the world.Weeks in 1993 etc., Perl etc. utilize the particle gun mediated method that gus gene, bar gene have been imported wheat, have set up the technical system of particle bombardment transformed wheat rataria.Yet agrobacterium-mediated transformation is one difficulty that wheat genetic transforms always.Cheng in 1997 etc. utilize agrobacterium-mediated transformation to change gus gene and npt II gene over to the wheat immature embryo callus first, have obtained the wheat transgenic plant, and to transfer-gen plant T
0Generation-T
2In generation, carried out Molecular Detection.Photosensitive grade of summer (1999), leaf make the country prosperous etc. (2001) utilize agrobacterium-mediated transformation to change foreign genes such as npt II, bar over to wheat, obtained transfer-gen plant.Zhou etc. utilize agrobacterium-mediated transformation to change the Roundup gene of antiweed over to wheat breed Bobwhite, have cultivated Roundup Ready transgenic wheat.Because some distinct advantages of agrobacterium-mediated transformation, fragment is clear and definite, copy number is low as shifting, easy expression, simple to operate and cost are low etc., and people are unremitting always to this research.Khanna in 2003 etc. make transformation efficiency reach 1.2-3.9% by making up super binary expression vector HK21 and add polyamine compounds in substratum; Hu etc. are selective marker with the anti-herbicide gene EPSPS that derives from Agrobacterium, are selective agent with careless fourth phosphine, and the EPSPS gene has been imported wheat breed Bobwhite, and transformation efficiency has reached 4.3%.Cheng etc. (2003) think that explant being carried out drying treatment behind the agroinfection can significantly improve T-DNA transhipment level and transformation efficiency.
It is acceptor material that 13-14 days the rataria in pollination back is adopted in wheat transgenic research mostly, generally adopt promotors such as Ubi, E35S and bar, nptII, selection markers genes such as EPSPS, hpt in the expression vector establishment, selective agent generally uses Bialaphos, Glufosinate, G418 and hygromycin etc., and the Agrobacterium fungus strain of utilization comprises ABI, Agl1, C58C1, LBA4404 and CP4 etc.Although agrobacterium-mediated transformation has been obtained certain progress; but utilize the Agrobacterium-mediated Transformation wheat still relatively more difficult at present; especially Agrobacterium to infect behind the wheat immature embryo phenomenon of callus browning death very serious; can not obtain the resistant calli and the regeneration plant of sufficient amount; be the bottleneck of wheat transgenic research, limited the large-scale production of transgenic wheat and the development of wheat functional genome research.Therefore, the problem of callus browning death is the key of wheat transgenic research behind the solution Agrobacterium-mediated Transformation wheat immature embryo.
Summary of the invention
The purpose of this invention is to provide a kind of Agrobacterium tumefaciens mediated transformed wheat rataria substratum.
Agrobacterium tumefaciens mediated transformed wheat rataria substratum provided by the present invention is the solid medium of molysite, sucrose, PAA and Dicamba of trace element, the MS minimum medium of the macroelement that contains the MS minimum medium, MS minimum medium, wherein the concentration of PAA is 0-1.0mg/L, the concentration of Dicamba is 2.0-3.0mg/L, and concentration of sucrose is 20.0-40.0g/L.
The concentration of PAA is preferably 0.5mg/L in the above-mentioned substratum, and the concentration of Dicamba is preferably 2mg/L, and concentration of sucrose is preferably 30g/L.
Another object of the present invention provides a kind of method of Agrobacterium tumefaciens mediated transformed wheat rataria.
The method of Agrobacterium tumefaciens mediated transformed wheat rataria provided by the present invention comprises the steps:
1) wheat immature embryo is inoculated on the above-mentioned substratum under 24-26 ℃ the culture temperature dark culturing 4-7 days;
2) infect the wheat immature embryo cultivated through step 1), carry out common cultivation with the purpose agrobacterium tumefaciens, the goal gene in the agrobacterium tumefaciens is imported wheat immature embryo.
Above-mentioned wheat immature embryo culture temperature is preferably 25 ℃, and above-mentioned agrobacterium tumefaciens is the agrobacterium tumefaciens that contains target gene.
Carrying out before agrobacterium tumefaciens transforms, the greenhouse that the wheat plant that is used for providing wheat immature embryo can be planted in temperature and be 15-25 ℃ grows.
Also comprise in the method for above-mentioned Agrobacterium tumefaciens mediated transformed wheat rataria will changing over to through the wheat immature embryo of cultivating altogether whenever going up and state the step of replenishing induced embryonic callus in the substratum that 10.0mg G418 and 250.0mg Pyocianil obtain in the substratum.
Also comprise the step that the embryo callus that will obtain carries out differentiation culture and takes root in the aforesaid method.
Utilize substratum of the present invention and method for transformation thereof, the inductivity of embryo callus and the pick-up rate of resistant plant all are improved.Wherein, the average inductivity of the embryo callus of wheat CB037 is 67.1%, and the average inductivity of the embryo callus of wheat stone 4185 is 56.4%, and the average inductivity of the embryo callus of wheat Bobwhite is 60.9%; The average pick-up rate of wheat CB037 resistant plant is 241%, and the average pick-up rate of wheat stone 4185 resistant plants is 204%, and the average pick-up rate of wheat Bobwhite resistant plant is 283%.
Description of drawings
Fig. 1 carries out culture effect after agrobacterium tumefaciens is infected for the conventional agrobacterium tumefaciens conversion method in offering according to sending the documents to the rataria of wheat CB037
Fig. 2 carries out culture effect after agrobacterium tumefaciens is infected for substratum of the present invention and method for transformation to the rataria of wheat CB037
Fig. 3 carries out culture effect after agrobacterium tumefaciens is infected for substratum of the present invention and method for transformation to the rataria of wheat stone 4185
The differentiation situation of the resistant calli of Fig. 4 after for the conventional agrobacterium tumefaciens conversion method transformed wheat CB037 in offering according to sending the documents
Fig. 5 is the differentiation situation of the resistant calli behind substratum of the present invention and the method for transformation transformed wheat CB037
Fig. 6 is the differentiation situation of the resistant calli behind substratum of the present invention and the method for transformation transformed wheat stone 4185
Embodiment
Experimental technique related among the following embodiment is ordinary method if no special instructions.
Embodiment 1, agrobacterium tumefaciens conversion method infect wheat immature embryo
One, culture medium preparation and sterilization
MSD
2P
0.5The a large amount of 100ml of substratum: 10 * MS, 100 * MS trace 10ml, 200 * MS molysite 5ml, sucrose 30g, be settled to 1000ml, regulate pH to 6.0 then, add plant gel (Phytagel) 2.4g again, above composition adopted 121 ℃ of high pressure moist heat sterilizations 20 minutes; When the substratum temperature after subject to sterilization is reduced to 65 ℃ of left and right sides, toluylic acid (PAA) 0.5mg and 3 behind the adding filtration sterilization, 6-two chloro-O-Anisic Acid (Dicamba) 2mg.
Compare MSD with traditional MS substratum
2P
0.5The distinguishing feature of substratum is whole organic compositions of having removed in the MS substratum, has replaced 2 with Dicamba, 4-D, and added PAA.
WCCP
0.5The a large amount of 10ml of substratum: 10 * MS, 100 * MS trace 1ml, 200 * MS molysite 0.5ml, maltose 40g, MgCl
20.75g, ethyl sulfonic acid (MES) 1.95g, be settled to 975ml, regulate pH to 5.4 then, above composition adopted 121 ℃ of high pressure moist heat sterilizations 20 minutes; Several compositions below the uniform mixing: 100 * MS VITAMIN 10ml, glutamine 0.5g, caseinhydrolysate 0.1g, glucose 10g, 100mg/ml vitamins C 1ml, 4-amino-3,5,6-trichloropyridine formic acid (Picloram) 2.2mg, Syringylethanone (AS) 39mg, be settled to 25ml, join behind the filtration sterilization in the substratum behind the above-mentioned autoclaving; When the substratum temperature after subject to sterilization is reduced to 65 ℃ of left and right sides, add PAA 0.5mg and Dicamba 2mg behind the filtration sterilization again.
The a large amount of 100ml of embryo callus division culture medium: 10 * MS, 100 * MS trace 10ml, 200 * MS molysite 5ml, 2,4-D 0.2mg, sucrose 30g, 100 * MS VITAMIN 10ml, be settled to 1000ml, regulate pH to 6.0 then, add agar 8g again, above composition adopted 121 ℃ of high pressure moist heat sterilizations 20 minutes; When the substratum temperature after subject to sterilization is reduced to 65 ℃ of left and right sides, carboxylic Bian penicillin 250mg and G41825mg behind the adding filtration sterilization.
Two, agrobacterium tumefaciens conversion method transformed wheat rataria
1, the pre-cultivation
It is in 15-25 ℃ the greenhouse that wheat CB037, wheat stone 4185 and wheat Bobwhite (China national germplasm resource bank) are planted in temperature, bloom rataria (heart-shaped phase of pollination back 13-14 days wheat CB037, wheat stone 4185 and wheat Bobwhite, size is 1.0-1.2mm), be seeded in above-mentioned MSD
2P
0.5On the substratum, 25 ℃ of dark conditions were cultivated 4-7 days down.
2, infect
The pBI121 plasmid that carries the nptII gene is changed among the agrobacterium tumefaciens C58C1 (Beijing Baeyer enlightening Bioisystech Co., Ltd), with the agrobacterium tumefaciens called after C58C1-121 that comprises the pBI121 plasmid that obtains.
Single colony inoculation of picking agrobacterium tumefaciens C58C1-121 is in the YEP liquid nutrient medium, and the 250rpm shaking culture is to OD under 28 ℃ of conditions
600=0.6-1.0.
With above-mentioned OD
600Value is collected for the centrifugal 10min of bacterium liquid 4500rpm under room temperature condition of the agrobacterium tumefaciens C58C1-121 of 0.6-1.0, and precipitation is resuspended in above-mentioned WCCP
0.5In the liquid nutrient medium, the rataria of above-mentioned pre-incubated wheat CB037, wheat stone 4185 and wheat Bobwhite is put into resuspended bacterium liquid respectively soaks 30min, during shake gently.
3, cultivate altogether
From the resuspended liquid of agrobacterium tumefaciens C58C1-121, take out the rataria of wheat CB037, wheat stone 4185 and wheat Bobwhite, place on the aseptic filter paper, cultivated altogether 2 days under 25 ℃ of dark conditions.
4, evoked callus
Respectively the rataria of wheat CB037, wheat stone 4185 and wheat Bobwhite is transferred to the MSD that replenishes 10.0mg G418 and 250.0mg Pyocianil in every liter of substratum
2P
0.5On the substratum, induced embryonic callus under 25 ℃ of dark conditions.The rataria of wheat CB037 after agrobacterium tumefaciens C58C1-121 infects the callus induction effect as shown in Figure 2, the callus induction effect is as shown in Figure 3 after agrobacterium tumefaciens C58C1-121 infects for the rataria of wheat stone 4185.The average inductivity of the embryo callus of wheat CB037 was 67.1% (inductivity of embryo callus refers to that the embryo callus number accounts for the percentage that agrobacterium tumefaciens transforms the rataria number), the average inductivity of the embryo callus of wheat stone 4185 is 56.4%, and the average inductivity of the embryo callus of wheat Bobwhite is 60.9%.
5, callus differentiation
The embryo callus of wheat CB037, wheat stone 4185 and wheat Bobwhite is transferred to respectively on the embryo callus division culture medium, and under the 15-25 ℃ of condition, differentiation culture was carried out in illumination every day in 12-14 hour.The differentiation situation of the resistant calli of the rataria of wheat CB037 after agrobacterium tumefaciens C58C1-121 infects as shown in Figure 5, the differentiation situation of the resistant calli of the rataria of wheat stone 4185 after agrobacterium tumefaciens C58C1-121 infects is as shown in Figure 6.
6, root culture
When the resistance regeneration bud height that differentiates from wheat CB037, wheat stone 4185 and wheat Bobwhite resistant calli reaches 2-3cm, forward to respectively on the root media, when adventive root length arrives 3-4cm, take out plant, thoroughly remove the root substratum, move in the soil and cultivate.The average pick-up rate of wheat CB037 resistant plant is 241%, and the average pick-up rate of wheat stone 4185 resistant plants is 204%, and the average pick-up rate of wheat Bobwhite resistant plant is 283%.
Comparative Examples 1
It is in 15-25 ℃ the greenhouse that wheat CB037, wheat stone 4185 and wheat Bobwhite are planted in temperature, conventional conversion method for agrobacterium tumefaciens (Cheng M et al. in offering according to sending the documents, Plant Physiol., 1997,115:971-980) rataria (heart-shaped phase, size is 1.0-1.2mm) to pollination back 13-14 days wheat CB037, wheat stone 4185 and the wheat Bobwhite of blooming transforms.The rataria of wheat CB037 carry out after conventional agrobacterium tumefaciens is infected culture effect as shown in Figure 1, the differentiation situation of its resistant calli is as shown in Figure 4.The result shows, after infecting according to the conventional agrobacterium tumefaciens conversion method in the above-mentioned document, the average inductivity of embryo callus is 5.6%, conventional agrobacterium tumefaciens conversion method infects wheat immature embryo browning problem of death existing (the David L P et al. of description in some documents that causes behind the wheat immature embryo, Physiological and Molecular Plant Pathology, 2002,60:59-69; Shrawat A K et al., Plant Biotechnology Journal, 2006,4:575-603).
Claims (8)
1. Agrobacterium tumefaciens mediated transformed wheat rataria substratum, it is composed as follows: the trace element of the macroelement of MS minimum medium, MS minimum medium, the molysite of MS minimum medium, concentration are that the sucrose of 30g/L, PAA and the concentration that concentration is 0.5mg/L are the Dicamba of 2.0mg/L; Described substratum is a solid medium.
2. the method for an Agrobacterium tumefaciens mediated transformed wheat rataria comprises the steps:
1) wheat immature embryo is inoculated in the described substratum of claim 1 under 24-26 ℃ the culture temperature dark culturing 4-7 days;
2) infect the wheat immature embryo handled through step 1), carry out common cultivation with the purpose agrobacterium tumefaciens, the target gene in the purpose agrobacterium tumefaciens is imported wheat immature embryo.
3. method according to claim 2 is characterized in that: the culture temperature of described wheat immature embryo is 25 ℃.
4. method according to claim 2 is characterized in that: contain target gene in the described purpose agrobacterium tumefaciens.
5. according to arbitrary described method among the claim 2-4, it is characterized in that: comprise also in the described method that it is the step that 15-25 ℃ greenhouse grows that the wheat plant that will be used for providing wheat immature embryo is planted in temperature.
6. method according to claim 5 is characterized in that: also comprise in the described method and will change the step of induced embryonic callus in the following substratum over to through the wheat immature embryo of cultivating altogether; Described substratum replenishes 10.0mg G418 in the described substratum of every liter of claim 1 and the 250.0mg Pyocianil obtains.
7. method according to claim 6 is characterized in that: comprise also in the described method that the embryo callus that will obtain carries out the step of differentiation culture.
8. method according to claim 7 is characterized in that: also be included in the step of taking root behind the described differentiation culture in the described method.
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