CN103409460A - Maize transformation method - Google Patents
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- CN103409460A CN103409460A CN201310367468XA CN201310367468A CN103409460A CN 103409460 A CN103409460 A CN 103409460A CN 201310367468X A CN201310367468X A CN 201310367468XA CN 201310367468 A CN201310367468 A CN 201310367468A CN 103409460 A CN103409460 A CN 103409460A
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Abstract
The invention relates to a maize transformation method which comprises the following steps: stripping young maize embryos for inducing callus; and infecting the callus by agrobacterium. The maize transformation method fulfills the purpose that the callus induced by the young maize embryos serves as an acceptor material for transformation for the first time, and ensures that the transformation rate can reach up to 5%; meanwhile, the maize callus with high quality and large amount can be obtained for subculture propagation, and can be easily available and also be supplied on a large scale, so that the requirement of maize transformation on the acceptor material can be fully met; the defect that the harvest condition of explants cannot be accurately predicted due to the limitation of weather conditions on field materials can be overcome, so that maize genetic transformation is not influenced by seasons, and the requirement on annual stable transformation can be met; in addition, the dependence on the young maize embryo in the prior art is cut, and maize does not need to be planted in a greenhouse and the like in non-maize-planting seasons, so that the cost for obtaining the acceptor material is greatly reduced.
Description
Technical field
The present invention relates to a kind of method of Plant Transformation, particularly relate to a kind of method of maize transformation callus.
Background technology
Corn is one of world's Three major grain crops, is also important industrial raw material and biological energy source substance, all occupies very important status in China and even global grain-production.In the factors that improves the corn per unit area yield, the effect of breeding of new variety accounts for 40%.Along with the tremendous development of Protocols in Molecular Biology, transgenic technology starts to play a significant role in the corn variety seed selection.Transgenic corns has become second largest genetically modified crops of global cultivated area that are only second to genetically engineered soybean.
Good receptor system is the important step that maize genetic transforms, be related to the acceptor material that suitable conversion is provided and transform after cell can be regenerated as the problems such as normal plant.In receptor system, how to improve the somatic regenerative power of corn particularly crucial.Good gene transformation receptor system should meet following condition: the regenerative power of (1) efficient stable; (2) acceptor material will have higher genetic stability: it is to import foreign gene to make it to integrate, express and heredity that plant gene transforms purpose, therefore, sets up the generation that receptor system will be avoided somatic variation as far as possible; (3) has stable explant source: for the acceptor transformed, will be easy to obtain and can supply in a large number, as rataria etc.; (4) to the selectivity antibiotic sensitive, namely when in selective medium, antibiotic concentration reaches certain value, can suppress growth, growth and the differentiation of non-transformed cell, and the vegetable cell transformed energy normal growth, division and differentiation owing to carrying antibiotic resistant gene obtains complete plant; (5) Agrobacterium is infected and susceptibility arranged but without anaphylaxis.
Up to now, in the genetic transformation of corn, the most effective acceptor material comprises the callus that callus that rataria, rataria are induced and cell suspension culture obtain, also have in addition the isolated protoplastis of callus, embryo callus or suspension cell line that mature embryo induces and shoot apical meristem etc., in recent years, many explants that directly utilize have been arranged as transformation receptor system the report of achieving success.Koziel etc. utilize the Bombardment-Mediated Transformation maize immature embryos to succeed.Zheng etc., with particle gun bombardment corn shoot tip meristem, import the Gus gene in corn, obtain transfer-gen plant.Also has document to have to utilize callus that coleoptile induces as explant material.But there is following defect in above-mentioned acceptor material:
(1) seasonality and poor stability: high to the explant material requirements, and explant obtains difficulty, due to the growth of explant, is subjected to simultaneously the restriction of weather condition, and the quality of material is difficult to control, and is difficult to meet the demand of anniversary stable conversion;
(2) comparatively difficult thereby the callus that coleoptile induces as the tissue broken up has more serious tissue characteristics to cause obtaining transfer-gen plant;
(3) transformation efficiency is on the low side, can't filter out the individual plant of good objective trait;
(4) utilize at present the callus that maize immature embryos induces to transform and have technology barrier as the acceptor material transformed.
Summary of the invention
A kind of method that the purpose of this invention is to provide maize transformation, the technological deficiency such as effectively overcome prior art seasonality, poor stability, tissue characteristics is serious, transformation efficiency is on the low side.
For achieving the above object, the invention provides a kind of method of maize transformation, comprising:
Strip maize immature embryos and carry out inducing of callus;
Agrobacterium is infected described callus.
On the basis of technique scheme, described rataria length is 0.6-1mm.
Preferably, the method for described maize transformation also comprises the described callus preculture 4-7 days after inducing.
Further, described Agrobacterium is infected described callus and is specially Agrobacterium and infects described callus infecting on substratum, and the described substratum that infects comprises tensio-active agent.
Alternatively, the method for described maize transformation also comprises the described callus after inducing was carried out to succeeding transfer culture 3 months at the most.
For achieving the above object, the present invention also provides a kind of method of producing the maize plant of stable conversion, comprising:
Strip maize immature embryos and carry out inducing of callus;
Agrobacterium is infected described callus;
Callus after infecting and described Agrobacterium are cultivated altogether;
Containing on the substratum of selective agent the callus of cultivating and selecting conversion;
The callus regeneration transformed is maize plant.
On the basis of technique scheme, described rataria length is 0.6-1mm.
Preferably, the method for described maize transformation also comprises the described callus preculture 4-7 days after inducing.
Further, described Agrobacterium is infected described callus and is specially Agrobacterium and infects described callus infecting on substratum, and the described substratum that infects comprises tensio-active agent.
Alternatively, the method for described maize transformation also comprises the described callus after inducing was carried out to succeeding transfer culture 3 months at the most.
Clone gene in the present invention, expression cassette, carrier (for example plasmid) but, albumen and protein fragments and the nucleus plant that transforms all the Application standard method produce.
The present invention is used in maize plant and expresses any goal gene.Goal gene can be Bar gene, disease-resistant gene or anti insect gene, or selection or evaluation mark, and contains the exercisable promotor of plant, coding region and termination subarea.Bar gene comprises to the AHAS gene of imidazolone or sulfonylurea herbicide tolerance, to the pat of careless fourth phosphine herbicide tolerant or bar gene, to the EPSPS gene of glyphosate herbicidal tolerance etc.Disease-resistant gene comprises the microbiotic synthase gene, pyrrolnitrin synthase gene for example, the resistant gene of plant origin etc.Anti insect gene comprises the bacillus thuringiensis killing gene.The goal gene enzyme relevant with bio-chemical pathway of also can encoding, the expression of this enzyme can change proterties important in food, feed, nutritional drugs and/or drug manufacture.Goal gene can be positioned on plasmid.The plasmid that applicable the present invention uses can contain an above goal gene and/or Agrobacterium can contain the different plasmids with different goal gene.
" corn " described in the present invention refers to Zea mays (Zea mays), and described method is to be transferred to maize calli with agriculture bacillus mediated goal gene, and the maize plant that is regenerated as afterwards conversion is basic.Method of the present invention is independent of Cultivar.
In the situation that existing, selective agent cultivates the maize calli transformed.Preferably, with phosphomannose isomerase (PMI) gene transformation, and the callus transformed is in the situation that seminose exists cultivates.In containing the substratum that seminose is selective agent, the maize calli of PMI gene transformation has growth vigor than unconverted maize calli.
It is involved in the present invention containing the transgenic plant of heterologous nucleic acids (namely containing the callus transformed according to method of the present invention) and seed and the offspring who produces by these transgenic plant.It is well known to a person skilled in the art that the callus culture of conversion is become to the method for useful Cultivar.Plant tissue Vitro Culture Techniques and whole plant regeneration technique are also known.Accordingly, described " seed " comprises the seed of these conversion of plants and the seed that the conversion of plant offspring produces.Described " plant " not only comprises the plant that transforms and regenerate, and also comprises by the conversion of method generation of the present invention and the offspring of aftergrowth.
Can be from the successful conversion of plant of screening the plant that method of the present invention produces.For the Plants and Seeds strain of development and improvement, the seed of Selection and screening aftergrowth of the present invention and progeny plant are with the nucleotide sequence sustainable existence of render transgenic and integration constantly.Therefore, needed transgenosis nucleotide sequence can be moved in (namely gradually oozing or mating) other genetic strain such as some original seed or commercial useful strain or kind.Gradually oozing the method that goal gene enters hereditary plant lines can realize by multiple technologies well known in the art, comprises by traditional breeding, protoplast fusion, nucleus and shifting and chromosome transfer.Breeding tactics is also well known in the art.The transgenic plant and the self-mating system that according to the present invention, obtain can be used for producing commercial valuable hybrid plant and farm crop.
The invention provides a kind of method of maize transformation, have the following advantages:
1, abundance.The method of maize transformation of the present invention, by obtaining the maize calli of better quality and quantity, is carried out the subculture expansion numerous, not only is easy to obtain and can supply in a large number, fully meets corn and transforms the demand to acceptor material.
2, stable.The method of maize transformation of the present invention has overcome the land for growing field crops material and limited by weather condition, can't estimate accurately the defect of explant results situation, is not subjected to seasonal the impact thereby make maize genetic transform, and meets the demand of anniversary stable conversion.
3, economy.The method of maize transformation of the present invention has been broken away from the dependence of prior art to maize immature embryos, without at non-corn planting season maize planting (as greenhouse), has saved greatly the cost that obtains acceptor material.
4, break through.The method of maize transformation of the present invention has realized utilizing the callus that maize immature embryos induces to transform as the acceptor material transformed first, and has guaranteed that transformation efficiency can reach 5%.
Below by drawings and Examples, technical scheme of the present invention is described in further detail.
The accompanying drawing explanation
Fig. 1 is that the recombinant cloning vector DBN01Ab-T of the method for maize transformation of the present invention builds schema;
Fig. 2 is that the recombinant expression vector DBN100053 of the method for maize transformation of the present invention builds schema;
Fig. 3 is the design sketch of maize transformation callus of the method for maize transformation of the present invention.
Embodiment
Below by specific embodiment, further illustrate the technical scheme of the method for maize transformation of the present invention.
The structure of the first embodiment, recombinant expression vector and recombinant expression vector transform Agrobacterium
1, build the recombinant cloning vector that contains goal gene
The Cry1Ab nucleotide sequence is connected into to cloning vector pGEM-T(Promega, Madison, USA, CAT:A3600) on, operation steps is undertaken by the product pGEM-T of Promega company carrier specification sheets, obtain recombinant cloning vector DBN01Ab-T, it builds flow process, and (wherein, Amp means ampicillin resistance gene as shown in Figure 1; F1 means the replication orgin of phage f1; LacZ is the LacZ initiator codon; SP6 is the SP6RNA polymerase promoter; T7 is the t7 rna polymerase promotor; Cry1Ab is Cry1Ab nucleotide sequence (SEQ ID NO:1); MCS is multiple clone site).
Then recombinant cloning vector DBN01Ab-T is transformed to intestinal bacteria T1 competent cell (Transgen by the heat shock method, Beijing, China, CAT:CD501), its hot shock condition is: 50 μ l intestinal bacteria T1 competent cells, 10 μ l plasmid DNA (recombinant cloning vector DBN01Ab-T), 42 ℃ of water-baths 30 seconds; Cultivate 1 hour (under the 200rpm rotating speed, shaking table shakes) for 37 ℃, on surface, scribble the IPTG(isopropylthio-β-D-galactoside) and the chloro-3-indoles-β of the bromo-4-of X-gal(5--D-galactoside) dull and stereotyped (the Tryptones 10g/L of LB of penbritin (100mg/L), yeast extract 5g/L, NaCl10g/L, agar 15g/L, adjust pH to 7.5 with NaOH) upper grow overnight.The picking white colony, in LB liquid nutrient medium (NaCl10g/L, penbritin 100mg/L, adjust pH to 7.5 with NaOH for Tryptones 10g/L, yeast extract 5g/L) under 37 ℃ of conditions of temperature overnight incubation.Alkaline process extracts its plasmid: by bacterium liquid centrifugal 1min under the 12000rpm rotating speed, remove supernatant liquor, the precipitation thalline is iced the solution I (25mM Tris-HCl, 10mM EDTA(ethylenediamine tetraacetic acid (EDTA)) of precoolings with 100 μ l, and 50mM glucose pH8.0) suspends; The solution II (0.2M NaOH, 1%SDS(sodium lauryl sulphate) that adds the new preparation of 150 μ l), pipe is put upside down 4 times, mixed, put 3-5min on ice; Add the solution III that 150 μ l are ice-cold (4M Potassium ethanoate, 2M acetic acid), fully mix immediately, place 5-10min on ice; Centrifugal 5min under 4 ℃ of temperature, rotating speed 12000rpm condition, add 2 times of volume dehydrated alcohols in supernatant liquor, mix rear room temperature and place 5min; Centrifugal 5min under 4 ℃ of temperature, rotating speed 12000rpm condition, abandon supernatant liquor, and precipitation is to dry after 70% washing with alcohol by concentration (V/V); Add 30 μ l to contain RNase(20 μ g/ml) TE(10mM Tris-HCl, 1mM EDTA, PH8.0) dissolution precipitation; In 37 ℃ of lower water-bath 30min of temperature, digestion RNA; In temperature-20 ℃, save backup.
The plasmid extracted, after ApaI and SpeI enzyme are cut evaluation, carries out sequence verification to positive colony, and result shows that the Cry1Ab gene order of inserting in recombinant cloning vector DBN01Ab-T is the nucleotide sequence shown in SEQ ID NO:1 in sequence table.
2, build the recombinant expression vector that contains goal gene
With restriction enzyme PvuI and KasI respectively enzyme cut recombinant cloning vector DBN01Ab-T and expression vector DBNBC-01(carrier framework: pCAMBIA2301(CAMBIA mechanism can provide)), the Cry1Ab gene order cut is inserted between the PvuI and KasI site of expression vector DBNBC-01, it is well-known to those skilled in the art utilizing conventional enzyme blanking method carrier construction, be built into recombinant expression vector DBN100053, it builds flow process (Kan: kanamycin gene as shown in Figure 2; RB: right margin; Ubi: corn Ubiquitin(ubiquitin) gene promoter (SEQ ID NO:2); Cry1Ab:Cry1Ab nucleotide sequence (SEQ ID NO:1); Nos: the terminator of rouge alkali synthetase gene (SEQ ID NO:3); PMI: Phophomannose isomerase gene (SEQ ID NO:4); LB: left margin).
Recombinant expression vector DBN100053 is transformed to intestinal bacteria T1 competent cell by the heat shock method, and its hot shock condition is: 50 μ l intestinal bacteria T1 competent cells, 10 μ l plasmid DNA (recombinant expression vector DBN100053), 42 ℃ of water-baths 30 seconds; Cultivate 1 hour (under the 200rpm rotating speed, shaking table shakes) for 37 ℃; Then containing LB solid plate (the Tryptones 10g/L of 50mg/L kantlex (Kanamycin), yeast extract 5g/L, NaCl10g/L, agar 15g/L, adjust pH to 7.5 with NaOH) above under 37 ℃ of conditions of temperature, cultivated 12 hours, the picking white colony, at LB liquid nutrient medium (Tryptones 10g/L, yeast extract 5g/L, NaCl10g/L, kantlex 50mg/L, adjust pH to 7.5 with NaOH) under 37 ℃ of conditions of temperature overnight incubation.Alkaline process extracts its plasmid.The plasmid of extraction is cut to rear evaluation with restriction enzyme PvuI and KasI enzyme, and by the positive colony evaluation of checking order, result show the nucleotides sequence of recombinant expression vector DBN100053 between PvuI and KasI site classify sequence table as in nucleotide sequence, i.e. Cry1Ab nucleotide sequence shown in SEQ ID NO:1.
3, recombinant expression vector transforms Agrobacterium
Oneself is transformed into to Agrobacterium LBA4404 (Invitrgen through building correct recombinant expression vector DBN100053 by the liquid nitrogen method, Chicago, USA, CAT:18313-015) in, its conversion condition is: 100 μ L Agrobacterium LBA4404s, 3 μ L plasmid DNA (recombinant expression vector); Be placed in liquid nitrogen 10 minutes, 37 ℃ of warm water bath 10 minutes; It is to cultivate 2 hours under the 200rpm condition in 28 ℃ of temperature, rotating speed that Agrobacterium LBA4404 after transforming is inoculated in the LB test tube, be applied on the LB flat board of kantlex (Kanamycin) of the Rifampin (Rifampicin) that contains 50mg/L and 100mg/L until grow positive monoclonal, its plasmid is cultivated and extracted to the picking mono-clonal, after with restriction enzyme HindIII and AscI enzyme, cutting, carry out enzyme and cut checking, result shows that recombinant expression vector DBN100053 structure is entirely true.
The second embodiment, proceed to the acquisition of the milpa of Cry1Ab nucleotide sequence
Stripping of maize immature embryos: corn variety is combined to 31(Z31) planting seed is in greenhouse or large Tanaka, 7-10 days after milpa grows up to pollination, when rataria length is 0.6-1mm, take and be no more than 1mm as good, strip rataria, at inducing culture (MS salt 4.3g/L, MS vitamin b6 usp, sucrose 30g/L, L-PROLINE 0.7g/L, 2,4-dichlorphenoxyacetic acid (2,4-D) 1.5mg/L, Silver Nitrate 0.85mg/L, MES (MES) 0.5g/L) on carry out inducing of callus, induction time was 2 weeks.Callus after inducing (500) is transferred to preculture substratum (MS salt 4.3g/L, MS vitamin b6 usp, sucrose 30g/L, L-PROLINE 0.7g/L, Silver Nitrate 0.85mg/L, MES (MES) 0.5g/L, 2,4-dichlorphenoxyacetic acid (2,4-D) 1.5mg/L) on, carry out preculture, the pre-incubated time is 4-7 days.To not carry out pre-incubated callus and carry out succeeding transfer culture, in order to retain standbyly, once, total Subculture Time is no more than 3 months to every 2 all subcultures.
The preparation of Agrobacterium bacterium liquid: picking contains the single bacterium colony of Agrobacterium of DBN100053, with the rifle head, being added with the upper line of the solid YP culture plate of spectinomycin (Spectinomycin) (yeast extract 5g, peptone 10g, sodium-chlor 5g, agar 15g, spectinomycin 50mg/L), under 28 ℃ of dark conditions, cultivate 2-3 days.Bacterial plaque on scraping YP culture plate, on new YP culture plate, cultivated again 1 day, scraping is cultivated the bacterium colony of 3-4 days, the liquid that infects that is suspended in 5ml (namely infects substratum (MS salt 4.3g/L, MS vitamin b6 usp, casein food grade 300mg/L, sucrose 68.5g/L, glucose 36g/L, Syringylethanone (AS) 40mg/L, 2,4-dichlorphenoxyacetic acid (2,4-D) 1mg/L, Selwet L-770.02%(v/v), pH5.3)) in, Agrobacterium bacterium liquid is mixed, its concentration is adjusted into to OD
660=0.4-0.6.
Agrobacterium is infected maize calli: in the centrifuge tube of 50ml, add the maize calli after the 5ml preculture, add simultaneously 2-3ml to infect liquid, then in the water-bath of 45 ℃, heated 5 minutes, again centrifuge tube is placed in and places 2 minutes on ice, after then infecting the liquid sucking-off, add 5ml Agrobacterium bacterium liquid, the vortex concussion kept flat 5 minutes by centrifuge tube after 3 minutes again; Infect after end the sucking-off of Agrobacterium bacterium liquid, with aseptic suction nozzle, Agrobacterium is thoroughly cleaned; Callus after infecting, from centrifuge tube, pouring out, is placed on three metafiltration paper of sky culture dish, then on callus, puts equally three metafiltration paper, and Agrobacterium bacterium liquid remaining on callus is thoroughly blotted.
Agrobacterium and maize calli are cultivated altogether: the callus that will blot Agrobacterium bacterium liquid is transferred to common culture medium (MS salt 4.3g/L, MS vitamin b6 usp, casein food grade 300mg/L, sucrose 30g/L, Syringylethanone (AS) 100mg/L, 2,4-dichlorphenoxyacetic acid (2,4-D) 1.5mg/L, L-PROLINE 0.7g/L, Silver Nitrate 0.85mg/L, Cys 0.3g/L, dithiothreitol (DTT) 0.4g/L, agar 8g/L, pH5.8) upper, callus and Agrobacterium were cultivated 3 days altogether.
The recovery of maize calli: the callus after cultivating is altogether transferred to recovery media (MS salt 4.3g/L, MS vitamin b6 usp, sucrose 30g/L, L-PROLINE 0.7g/L, 2,4-dichlorphenoxyacetic acid (2,4-D) 1.5mg/L, Silver Nitrate 0.85mg/L, MES (MES) 0.5g/L, cephamycin 0.25g/L, agar 8g/L) on, recovered 7 days, and take and eliminate Agrobacterium and provide decubation as callus.
The screening of maize calli: after decubation finishes, callus is transferred to screening culture medium (MS salt 4.3g/L, MS vitamin b6 usp, sucrose 5g/L, L-PROLINE 0.7g/L, 2,4-dichlorphenoxyacetic acid (2,4-D) 1.5mg/L, Silver Nitrate 0.85mg/L, MES 0.5g/L, cephamycin 0.25g/L, seminose 12.5g/L, agar 8g/L, pH5.8) upper, cause the callus transformed selective growth.In screening minute two stages, in each 2 week of stage, after screening, obtain resistant calli (100).
Maize calli regeneration plant: resistant calli is transferred to MS division culture medium (MS salt 4.3g/L, MS vitamin b6 usp, sucrose 30g/L, furfuryladenine 2mg/L, cephamycin 0.25g/L, seminose 5g/L, agar 8g/L, pH5.8) upper, cultivate differentiation 1 month under 25 ℃; It is upper that out seedling of differentiation is transferred to MS root media (MS salt 2.15g/L, MS vitamin b6 usp, casein food grade 300mg/L, sucrose 30g/L, indole-3-acetic acid 1mg/L, agar 8g/L, pH5.8), is cultured to about 10cm under 25 ℃ high, moves to hot-house culture.In greenhouse, cultivated 16 hours every day under 28 ℃, then cultivated 8 hours under 20 ℃, can obtain transfer-gen plant.
The 3rd embodiment, with TaqMan checking, proceed to the milpa of Cry1Ab nucleotide sequence
Get the about 100mg of blade of the milpa that proceeds to the Cry1Ab nucleotide sequence as sample, extract its genomic dna with the DNeasy Plant Maxi Kit of Qiagen, by the Taqman fluorescence probe quantitative PCR method, detect the copy number of Cry1Ab gene.Simultaneously with the wild-type milpa in contrast, detect according to the method described above analysis.3 repetitions are established in experiment, average.
The concrete grammar that detects the Cry1Ab gene copy number is as follows:
Step 11, get the milpa that proceeds to the Cry1Ab nucleotide sequence and each 100mg of blade of wild-type milpa, in mortar, be ground into homogenate with liquid nitrogen respectively, each sample is got 3 repetitions;
The DNeasy Plant Mini Kit of step 12, use Qiagen extracts the genomic dna of above-mentioned sample, and concrete grammar is with reference to its product description;
Step 13, use NanoDrop2000(Thermo Scientific) measure the genomic dna concentration of above-mentioned sample;
The genomic dna concentration of step 14, the above-mentioned sample of adjustment is to the same concentration value, and the scope of described concentration value is 80-100ng/ μ l;
Step 15, adopt the Taqman fluorescence probe quantitative PCR method to identify the copy number of sample, using through the sample of identifying the known copy number as standard substance, with the sample of wild-type milpa in contrast, 3 repetitions of each sample, get its mean value; Fluorescence quantification PCR primer and probe sequence are respectively:
Following primer and probe are used for detecting the Cry1Ab nucleotide sequence:
Primer 1(CF1): CGTCAGCGGTTTATCGGAAG is as shown in SEQ ID NO:5 in sequence table;
Primer 2 (CR1): AGGGACGTTATTGTTCTGTGGC is as shown in SEQ ID NO:6 in sequence table;
Probe 1(CP1): TGGCACCGTGGACTCACTCGATGA is as shown in SEQ ID NO:7 in sequence table;
The PCR reaction system is:
Described 50 * primer/probe mixture comprises each 45 μ l of every kind of primer of 1mM concentration, probe 50 μ l and the 860 μ l1 * TE damping fluid of 100 μ M concentration, and, at 4 ℃, be housed in the amber test tube.
The PCR reaction conditions is:
Utilize SDS2.3 software (Applied Biosystems) analytical data.
Experimental result shows, the positive plant that the Cry1Ab nucleotide sequence is incorporated in the genome of the milpa detected is 25 strains, and transformation efficiency can reach 5%, as table 1 and shown in Figure 3.
Table 1, the experimental result of maize calli as the transformation receptor material of usining
| Initial callus quantity (individual) | Kanamycin-resistant callus tissue quantity (individual) | Positive plant number (strain) | Transformation efficiency (%) |
| 500 | 100 | 25 | 5 |
In sum, the method for maize transformation of the present invention has realized utilizing the callus that maize immature embryos induces to transform as the acceptor material transformed first, and has guaranteed that transformation efficiency can reach 5%; By obtaining the maize calli of better quality and quantity, carry out the subculture expansion numerous simultaneously, not only be easy to obtain and can supply in a large number, fully meet corn and transform the demand to acceptor material; And overcome the land for growing field crops material and limited by weather condition, can't estimate accurately the defect of explant results situation, be not subjected to seasonal the impact thereby make maize genetic transform, met the demand of anniversary stable conversion; Break away from addition the dependence of prior art to maize immature embryos, without at non-corn planting season maize planting (as greenhouse), saved greatly the cost that obtains acceptor material.
It should be noted last that, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although with reference to preferred embodiment, the present invention is had been described in detail, those of ordinary skill in the art is to be understood that, can modify or be equal to replacement technical scheme of the present invention, and not break away from the spirit and scope of technical solution of the present invention.
Claims (10)
1. the method for a maize transformation, is characterized in that, comprising:
Strip maize immature embryos and carry out inducing of callus;
Agrobacterium is infected described callus.
2. the method for maize transformation according to claim 1, is characterized in that, described rataria length is 0.6-1mm.
3. according to the method for claim 1 or 2 described maize transformation, it is characterized in that, also comprise the described callus preculture 4-7 days after inducing.
4. the method for maize transformation according to claim 1, is characterized in that, described Agrobacterium is infected described callus and is specially Agrobacterium and infects described callus infecting on substratum, and the described substratum that infects comprises tensio-active agent.
5. the method for maize transformation according to claim 1, is characterized in that, also comprises the described callus after inducing was carried out to succeeding transfer culture 3 months at the most.
6. a method of producing the maize plant of stable conversion, is characterized in that, comprising:
Strip maize immature embryos and carry out inducing of callus;
Agrobacterium is infected described callus;
Callus after infecting and described Agrobacterium are cultivated altogether;
Containing on the substratum of selective agent the callus of cultivating and selecting conversion;
The callus regeneration transformed is maize plant.
7. the method for the maize plant of production stable conversion according to claim 6, is characterized in that, described rataria length is 0.6-1mm.
8. according to the method for the maize plant of the described production stable conversion of claim 6 or 7, it is characterized in that, also comprise the callus preculture 4-7 days after described inducing.
9. the method for the maize plant of production stable conversion according to claim 6, it is characterized in that, described Agrobacterium is infected described callus and is specially Agrobacterium and infects described callus infecting on substratum, and the described substratum that infects comprises tensio-active agent.
10. the method for the maize plant of production stable conversion according to claim 6, is characterized in that, also comprises the described callus after inducing was carried out to succeeding transfer culture 3 months at the most.
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| CN107119069A (en) * | 2017-05-10 | 2017-09-01 | 扬州大学 | A kind of corn inbred line stem apex live body method for transformation |
| CN108441512A (en) * | 2018-04-20 | 2018-08-24 | 刘寒冬 | A kind of corn genetic transformation method |
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| EP3090046A4 (en) * | 2013-12-31 | 2017-06-21 | Dow AgroSciences LLC | Novel maize ubiquitin promoters |
| CN104988178A (en) * | 2015-06-29 | 2015-10-21 | 吉林省农业科学院 | Genetic transformation method utilizing agrobacterium tumefaciens to infect maize immature embryos |
| CN106520661A (en) * | 2016-10-12 | 2017-03-22 | 北京大北农科技集团股份有限公司 | Corn transforming method |
| CN107119069A (en) * | 2017-05-10 | 2017-09-01 | 扬州大学 | A kind of corn inbred line stem apex live body method for transformation |
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