CN102952823B - Wheat genetic transformation method - Google Patents
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- CN102952823B CN102952823B CN201210505827.9A CN201210505827A CN102952823B CN 102952823 B CN102952823 B CN 102952823B CN 201210505827 A CN201210505827 A CN 201210505827A CN 102952823 B CN102952823 B CN 102952823B
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Abstract
The invention discloses a wheat genetic transformation method, and particularly relates to a method for increasing wheat genetic transformation efficiency through antibiotic gradient screening. The method comprises the following steps of: (1) carrying out pre-culturing on wheat embryonic calluses; (2) introducing expression vectors comprising target genes and hygromycin resistant genes into the embryonic calluses through a gene gun method; (3) recovering culture; (4) culturing on a screen culture medium for 35-55 days, wherein 35-45 mg.L<-1> of hygromycin is included in the screening culture medium; (5) culturing on a differentiation culture medium including 35-45 mg.L<-1> of hygromycin for 10-20 days to obtain the embryonic calluses for producing bud points; (6) culturing on a differentiation culture medium including 15-25 mg.L<-1> of hygromycin for 30-60 days to obtain plantlets; and (7) culturing on a rooting culture medium until roots are grown. The wheat genetic transformation method has the advantages of simplicity and easiness in operation, high transformation efficiency, good growth state of wheat plantlets obtained after transformation and extremely great actual application value and market promotion prospect.
Description
Technical field
The present invention relates to a kind of Efficiency of Wheat Transformation method, relate in particular to a kind of method that increases genetic transformation efficiency of wheat by the screening of microbiotic gradient.
Background technology
Wheat is one of most important food crop in the world, is the second largest food crop that are only second to paddy rice, all over the world, is all widely cultivated.Wheat is the farm crop that China people are rely and made a living, and along with the increase of China's population and the raising of living standards of the people, the yield and quality of wheat has also been proposed to more and more higher requirement.Traditional conventional breeding can not meet the requirement to the variety and quality of wheat, the restriction of conventional breeding has been broken in the development of transgenic technology, successfully in the crops such as cotton, corn, soybean, rape, cultivate high-yield variety, created good economic benefit and social benefit.Wheat is because transformation efficiency is lower, the more difficult acquisition of transgenic line, thereby to compare molecular breeding relative backward with other crop.
The method for transformation of wheat mainly contains pollen tube passage method, particle bombardment, agrobacterium co-cultivation, Electroporation method and PEG method at present.Due to wheat protoplast regeneration difficulty, so limited the application of Electroporation method and PEG conversion method.
The genetic transformation of pollen tube channel mediation is, after plant pollination, exogenous dna fragment is imported to blastular by pollen tube channel, thereby is incorporated in zygote genome, then develops into seed.Pollen tube passage method is simple, but transformation generation character variation is complicated, and later stage screening operation is very difficult, thereby is never widely used.Agriculture bacillus mediated Efficiency of Wheat Transformation is a difficult problem of vegitabilia's research always.People have done deeply research widely to affecting the factor of agriculture bacillus mediated Efficiency of Wheat Transformation, comprise wheat genotypes, explant source, preculture time, infect and be total to incubation time, Syringylethanone, agrobacterium strains, carrier and selective agent etc. and all can affect transformation efficiency.Agrobacterium co-cultivation has the low copy of transgenosis, inheritance stability, can transform relatively large fragment DNA and the advantage such as with low cost, but exist and to be subject to genotype restriction, to problems such as tissue culture technique dependency are strong, transformation frequency is not high and initial explant is single, thereby application is subject to certain restrictions.
Particle bombardment is widely used a kind of method for transformation during wheat transforms.Via Particle Bombardment Transformation method is that the metal particle by high-speed motion makes to be attached to its surperficial nucleic acid molecule through the cell walls of acceptor, and the DNA molecular discharging, and random integration is in Plant Genome, then by the tissue culture technique plant that regenerates.
Summary of the invention
The object of this invention is to provide a kind of Efficiency of Wheat Transformation method, relate in particular to a kind of method that increases genetic transformation efficiency of wheat by the screening of microbiotic gradient.
Efficiency of Wheat Transformation method provided by the invention, comprises the steps:
(1) wheat callus is carried out to preculture;
(2) expression vector that contains goal gene is imported to the embryo callus of completing steps (1) by particle bombardment; Described expression vector contains hygromycin gene;
(3) embryo callus of completing steps (2) is carried out to renewal cultivation;
(4) get the embryo callus of completing steps (3), in screening culture medium, cultivate 35-55 days (can be 35-45 days, 45-55 days, 35 days, 45 days or 55 days); In described screening culture medium, contain 35-45mgL
-1(can be 37-43mgL
-1, 37-40mgL
-1, 40-43mgL
-1, 37mgL
-1, 40mgL
-1or 43mgL
-1) Totomycin;
(5) get the embryo callus that step (4) obtains, containing 35-45mgL
-1(can be 37-43mgL
-1, 37-40mgL
-1, 40-43mgL
-1, 37mgL
-1, 40mgL
-1or 43mgL
-1) cultivate 10-20 days (can be 10-15 days, 15-20 days, 10 days, 15 days or 20 days) on the division culture medium of Totomycin, obtain producing the embryo callus of bud point;
(6) get the embryo callus of the generation bud point that step (5) obtains, containing 15-25mgL
-1(can be 17-23mgL
-1, 17-20mgL
-1, 20-23mgL
-1, 17mgL
-1, 20mgL
-1or 23mgL
-1) cultivate 30-60 days (can be 30-47 days, 47-60 days, 30 days, 47 days or 60 days) on the division culture medium of Totomycin, obtain seedling;
(7) get the seedling that step (6) obtains, on root media, be cultured to and take root.
In described step (1), the substratum that described preculture adopts can be height and oozes substratum.
In described step (1), described pre-incubated condition is: 25 ℃ of dark culturing 4 hours.
In described step (2), described expression vector can be plant expression vector.The carrier that described expression vector specifically can be pUN1301 carrier, the pUN1301 carrier of take is skeleton, in the multiple clone site of pUN1301 carrier, insert carrier, pCAMBIA1301 carrier that foreign gene obtains, carrier that the pCAMBIA1301 carrier of take is skeleton or insert in the multiple clone site of pCAMBIA1301 carrier vector the carrier that foreign gene obtains.In described step (2), described goal gene can be gus gene.In described step (2), the parameter of described particle bombardment can be: the concentration of described expression vector is 1ug/ul, adopts 1.0 μ m bronzes and 1100psi can split film.
The construction process of described pUN1301 carrier is as follows: take pCAMBIA1301 carrier as skeleton carrier, in the UbiPro(promotor shown in the sequence 1 of its multiple clone site insertion sequence table) and the sequence 2 of sequence table shown in Noster(terminator).
The construction process of described pUN1301 carrier is specific as follows: take pCAMBIA1301 carrier as skeleton carrier, small segment between BamHI and HindIII restriction enzyme site is replaced for the UbiPro(promotor shown in the sequence 1 of sequence table), the small segment between SacI and EcoRI restriction enzyme site is replaced for the Noster(terminator shown in the sequence 2 of sequence table).
In described step (3), the substratum that described renewal cultivation adopts is that height oozes substratum.
In described step (3), the condition of described renewal cultivation is: 25 ℃ of dark culturing 12 hours.
In described step (4), the substratum of described screening culture medium for adding Totomycin to obtain in embryo callus subculture inducing culture.
In described step (4), the condition of described cultivation is: 25 ℃ of dark culturing, the screening culture medium that every 10-15 days more renews subculture.
In described step (5): described division culture medium is embryo callus subculture division culture medium.
In described step (5), the condition of described cultivation is: 25 ℃, light dark period is 12 hours/12 hours.
In described step (6): described division culture medium is embryo callus subculture division culture medium.
In described step (6), the condition of described cultivation is: 25 ℃, light dark period is 12 hours/12 hours.
In described step (7), the condition of described cultivation is: 25 ℃, light dark period is 12 hours/12 hours.
Described wheat can be cultivation or Wild Wheats kind, strain, breeding material or intermediate materials, specifically can be wheat breed " capital winter No. 1 " or wheat breed " spend No. 9 in capital ".
In described step (1), described wheat callus can be and expands numerous wheat callus obtaining; The numerous method of described expansion is as follows: by wheat callus on embryo callus subculture inducing culture 25 ℃ dark culturing 4-5 month, every 2 weeks subcultures once, all adopt embryo callus subculture proliferated culture medium during subculture.
The preparation method of arbitrary described embryo callus subculture inducing culture is as follows above: in MS substratum, add 2,4 dichlorophenoxyacetic acid, glutamine and caseinhydrolysate, the concentration that makes 2,4 dichlorophenoxyacetic acid is 1-2mgL
-1(as 1.5-2mgL
-1, 1.5mgL
-1or 2mgL
-1), the concentration of glutamine is 200-700mgL
-1(as 300-700mgL
-1, 300-500mgL
-1, 500-700mgL
-1, 500mgL
-1, 300mgL
-1or 700mgL
-1), the concentration of caseinhydrolysate is 500-700mgL
-1(as 300-700mgL
-1, 300-500mgL
-1, 500-700mgL
-1, 500mgL
-1, 300mgL
-1or 700mgL
-1).
The preparation method of arbitrary described embryo callus subculture proliferated culture medium above: add 2,4 dichlorophenoxyacetic acid, glutamine and caseinhydrolysate in MS substratum, the concentration that makes 2,4 dichlorophenoxyacetic acid is 1-2mgL
-1(as 1-1.5mgL
-1, 1.5-2mgL
-1, as 1mgL
-1, 1.5mgL
-1or 2mgL
-1), the concentration of glutamine is 200-700mgL
-1(as 300-700mgL
-1, 300-500mgL
-1, 500-700mgL
-1, 500mgL
-1, 300mgL
-1or 700mgL
-1), the concentration of caseinhydrolysate is 500-700mgL
-1(as 300-700mgL
-1, 300-500mgL
-1, 500-700mgL
-1, 500mgL
-1, 300mgL
-1or 700mgL
-1).
The preparation method that arbitrary described height oozes substratum above: add N.F,USP MANNITOL in described embryo callus subculture inducing culture, the concentration that makes N.F,USP MANNITOL is 0.3-0.8molL
-1(as 0.3-0.4molL
-1, 0.4-0.8molL
-1, 0.3molL
-1, 0.4molL
-1or 0.8molL
-1).
The preparation method of arbitrary described screening culture medium above: add Totomycin in described embryo callus subculture inducing culture, the concentration that makes Totomycin is 35-45mgL
-1(can be 37-43mgL
-1, 37-40mgL
-1, 40-43mgL
-1, 37mgL
-1, 40mgL
-1or 43mgL
-1).
The preparation method of arbitrary described embryo callus subculture division culture medium above: add 6-glycosyl aminopurine and caseinhydrolysate in MS substratum, the concentration that makes 6-glycosyl aminopurine is 0.5-4mgL
-1(as 1.5-3mgL
-1, 1.5-2mgL
-1, 2-3mgL
-1, 1.5mgL
-1, 2mgL
-1or 3mgL
-1), the concentration of caseinhydrolysate is 500-700mgL
-1(as 200-500mgL
-1, 500-700mgL
-1, 500mgL
-1, 200mgL
-1or 700mgL
-1).
The preparation method of arbitrary described root media adds α-naphthaleneacetic acid in MS substratum above, and the concentration that makes α-naphthaleneacetic acid is 0.5-1.0mgL
-1(as 0.5mgL
-1).
Arbitrary described method all can be used for wheat breeding above.
The preparation method of arbitrary described MS substratum is specially above: according to the add-on of table 1, by each component, water-soluble and water is settled to 1L.
The present invention is to provide a kind of method of utilizing gradient sieve method to improve the transformation efficiency of Bombardment-Mediated Transformation wheat.Operation is simple in invention, and transformation efficiency is high, and after transforming, resulting transgenic wheat growth of seedling is in good condition, has very large actual application value and marketing prospect.
Accompanying drawing explanation
Fig. 1 is the photo of cultivating the callus obtaining after 4-5 month on embryo callus subculture inducing culture; A is photo; B is the local photo amplifying.
Fig. 2 is the high photo of cultivating callus after 4 hours on substratum that oozes.
Fig. 3 is the photo of cultivating resistant calli after 45 days in screening culture medium.
Fig. 4 cultivates the photo that produces afterwards the embryo callus of bud point for 15 days in substratum first; A is photo; B is the local photo amplifying.
Fig. 5 is the photo of callus while cultivating 47 days in substratum second.
Fig. 6 is the photo of the seedling in rooting process.
Fig. 7 is the photo of transplanting in plant strain growth process after flowerpot.
Fig. 8 is the photo of transplanting to solid plant after flowerpot.
Fig. 9 is the photo of cultivating 3-4 callus during week in substratum second or substratum first.
Figure 10 is the photo after GUS dyeing.
Figure 11 is the photo of embodiment 5.
Figure 12 is the structural representation of pCAMBIA1301 carrier.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, be ordinary method, concrete steps can be referring to: < < Molecular Cloning:ALaboratory Manual > > (Smabrook, J., Ressule L., David W., Molecular Cloning:A Laboratory Manual, 3
rdedition, 2001, NY, Cold Spring Harbor).Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment, results averaged for three times.
Wheat breed " capital winter No. 1 ": reference: the new agriculture > > of < <; Exercise question: New Winter Wheat Variety---the capital winter No. 1; Author: Zhai Hui, phase volume: 09 phase in 2003: 44 pages.
Wheat breed " spend No. 9 in capital ": reference: < < wheat crops journal > >, exercise question: spend No. 9 in the precocious New Winter Wheat Variety-capital of high yield and high quality; Author: blue or green brave single Fu Hua Zhang Liping Zhao of Zhao Ke of Liu Jianping Zhang Liquan field Li Pingsu Changping; Phase volume: 2008,28 phases 5 volumes: 921 pages.
The preparation of embodiment 1, substratum
The pH value of each substratum of the present embodiment is 5.8, has prepared rear 120 ℃ of autoclavings 20 minutes.
One, the preparation of MS substratum
Each component that every liter of MS substratum adds and add-on thereof are in Table 1.
The content of each solute in every liter of MS substratum of table 1
| Add-on (g) | |
| Saltpetre (KNO 3) | 1.90 |
| Ammonium nitrate (NH 4NO 3) | 1.65 |
| Potassium primary phosphate (KH 2PO 4) | 0.17 |
| Magnesium sulfate heptahydrate (MgSO 4·7H 2O) | 0.37 |
| Calcium dichloride dihydrate (CaCl 2·2H 2O) | 0.44 |
| Potassiumiodide (KI) | 0.00083 |
| Boric acid (H 3BO 3) | 0.0062 |
| Four water manganous sulfate (MnSO 4·4H 2O) | 0.0223 |
| Zinc Sulphate Heptahydrate (ZnSO 4·7H 2O) | 0.0086 |
| Sodium Molybdate Dihydrate (Na 2MoO 4·2H 2O) | 0.00025 |
| Cupric sulfate pentahydrate (CuSO 4·5H 2O) | 0.000025 |
| CoCL2 6H2O (CoCl 2·6H 2O) | 0.000025 |
| Disodium ethylene diamine tetraacetate (Na 2·EDTA) | 0.0373 |
| Iron vitriol (FeSO 4·7H 2O) | 0.0278 |
| Glycine (Glycine) | 0.002 |
| Vitamin (VB1) | 0.0001 |
| Pyridoxine hydrochloride (VB6) | 0.0005 |
| Nicotinic acid (VB5) | 0.0005 |
| Maltose (Maltose) | 30 |
| Plant gel Gelzan(Sigma company) | 2.5 |
The preparation method of MS substratum: water-soluble and water is settled to 1L by each component according to the add-on of table 1, adjust pH to 5.8,120 ℃ of autoclavings 20 minutes.
The MS substratum of preparing by step 1 is as basic medium, other substratum in preparation steps two.
Two, the preparation of other substratum
The preparation method of embryo callus subculture inducing culture: (be commonly called as 2,4-D), glutamine and caseinhydrolysate, the concentration that makes 2,4 dichlorophenoxyacetic acid is 2mgL to add 2,4 dichlorophenoxyacetic acid in MS substratum
-1, glutamine concentration be 500mgL
-1, caseinhydrolysate concentration be 500mgL
-1.
The preparation method of embryo callus subculture proliferated culture medium: add 2,4 dichlorophenoxyacetic acid, glutamine and caseinhydrolysate in MS substratum, the concentration that makes 2,4 dichlorophenoxyacetic acid is 1mgL
-1, glutamine concentration be 500mgL
-1, caseinhydrolysate concentration be 500mgL
-1.
Height oozes the preparation method of substratum: in embryo callus subculture inducing culture, add N.F,USP MANNITOL, the concentration that makes N.F,USP MANNITOL is 0.4molL
-1.
The preparation method of screening culture medium: add Totomycin in embryo callus subculture inducing culture, the concentration that makes Totomycin is 40mgL
-1.
The preparation method of embryo callus subculture division culture medium: add 6-glycosyl aminopurine (being commonly called as kinetin KT) and caseinhydrolysate in MS substratum, the concentration that makes 6-glycosyl aminopurine is 2mgL
-1, caseinhydrolysate concentration be 500mgL
-1.
The preparation method of root media adds α-naphthaleneacetic acid (being commonly called as growth hormone NAA) in MS substratum, and the concentration that makes α-naphthaleneacetic acid is 0.5mgL
-1.
The structure of embodiment 2, recombinant plasmid
One, the structure of pUN1301 carrier
With pCAMBIA1301 carrier (as shown in the sequence 4 of sequence table; Structural representation is shown in Figure 12; Purchased from Christian Dior bio tech ltd, Shanghai, catalog number BDRH209) be skeleton carrier, small segment between BamHI and HindIII restriction enzyme site is replaced for the UbiPro(promotor shown in the sequence 1 of sequence table), the small segment between SacI and EcoRI restriction enzyme site is replaced for the Noster(terminator shown in the sequence 2 of sequence table).
Two, the structure of TaCYP-pUN1301 carrier
Small segment between pUN1301 carrier KpnI and SacI restriction enzyme site is replaced with to the TaCYP gene shown in the sequence 3 of sequence table (TaCYP gene is the gene relevant to wheat plant types, and overexpression can make plant downgrade).
The genetic transformation of embodiment 3, wheat
Adopt the substratum of embodiment 1 preparation to carry out embodiment 3.
One, the preparation of wheat callus
1, the immature seed of 2 weeks after getting wheat breed " capital winter No. 1 " and blooming, peel off bran sheet, being placed in 70%(volume ratio) ethanol aqueous solution soaks 30 seconds, with sterile distilled water, rinse 2 times, then be placed in and proceed to the 0.1g/100mL mercuric chloride aqueous solution and soak 10 minutes (sterilization), with sterile distilled water, rinse after 4-5 time, then operation surgical forceps is chosen embryo, shield towards on be placed on embryo callus subculture inducing culture, 25 ℃ within dark culturing 4-5 month, (every 2 weeks subcultures once, during subculture, all adopt embryo callus subculture proliferated culture medium), obtain particle less, fine and close frangible, pale yellow vivid callus (Fig. 1 is shown in by photo).
2, the height that is placed in of getting 2.5 centimetres of diameters the callus obtaining from step 1 oozes substratum central authorities, and each culture dish is put 100 left and right callus, 25 ℃ of dark culturing 4 hours (Fig. 2 is shown in by photo).
Two, Wheat Transformation Efficiency By Particle Bombardment callus
The callus that pUN1301 carrier is obtained by particle gun implant steps one.Bombarding conditions: the concentration of pUN1301 carrier is 1 μ g/ μ l; Adopting diameter is the bronze of 1.0 μ m, the split film of 1100psi; Target material to the distance that can split film is 9 centimetres.
Three, transform the gradient screening of rear wheat callus
1, first group of processing
(1) callus of completing steps two is placed in to height and oozes on substratum, 25 ℃ of dark culturing 12 hours.
(2) callus of completing steps (1) is placed in screening culture medium, 25 ℃ of dark culturing 45 days, every screening culture medium more renewing for 15 days subculture, the pale yellow callus vivid and molecular marker for increased proliferation of color is resistant calli (Fig. 3 is shown in by photo), and color becomes dun and the callus of not breeding is non-resistance callus.
(3) resistant calli step (2) being obtained is placed in the substratum first (preparation method of substratum first: add Totomycin at embryo callus subculture division culture medium, the concentration that makes Totomycin is 40mgL
-1) upper, cultivate 15 days, obtain producing the embryo callus (Fig. 4 is shown in by photo) of bud point.Culture condition: 25 ℃, light dark period is 12 hours/12 hours.
(4) embryo callus of generation bud point step (3) being obtained is placed in the substratum second (preparation method of substratum second: add Totomycin at embryo callus subculture division culture medium, the concentration that makes Totomycin is 20mgL
-1) upper, cultivate 47 days, the substratum second the subculture that within every 15 days, more renew, bud point develops into seedling.Culture condition: 25 ℃, light dark period is 12 hours/12 hours.Cultivate the photo of 3-4 during week and see Fig. 9 A, most bud points can normal development become seedling.Fig. 5 is shown in by the photo of cultivating while finishing.
(5) seedling of 2 centimetres of left and right that step (4) obtained is placed on root media, and being cultured to little seedling rooting and root length is 10 centimetres of left and right (seedling in rooting process is shown in Fig. 6), opens container closure film, hardening 2-3 days.Culture condition is: 25 ℃, light dark period is 12 hours/12 hours.
Obtain altogether the plant that 149 strains survive.Seedling rate is 49%.The number that survives plant that seedling rate=step (5) obtains/for embryo callus number * 100% of the generation bud point of step (4).
(6) plantlet of transplant step (5) being obtained Nutrition Soil and the vermiculite of mass mixing (be equipped with etc.) in flowerpot is cultivated in phytotron.Culture condition is: 21 ℃ ± 5 ℃, light dark period is 16 hours/8 hours.Fig. 7 is shown in by photo in plant strain growth process, and Fig. 8 is shown in by the solid photo of plant.Wheat plant is energy normal growth all, blossoms and bears fruit, and breeds smoothly the next generation.
(7) by GUS, dye and identify genetic transformation efficiency
GUS dye liquor (pH 7.0) is comprised of water and solute; Solute and the concentration in GUS dye liquor thereof are as follows: 100mM sodium phosphate, 10mM EDTA, 0.5%(volume ratio) Sheng Ruitai Science and Technology Ltd. in Triton X-100,0.5mM yellow prussiate of potash, the 0.5mM Tripotassium iron hexacyanide and 1mM X-Gluc(Beijing.
Get the root of cultivating the plant of 1 month in step (6), in GUS dye liquor, in 25 ℃ of soaked overnight, show that blue plant has GUS activity, positive plant.
In the plant that 149 strains survive, the positive plant of 31 strain, genetic transformation rate is 20.8%.In the plant that 149 strains survive, a little less than root shows, Figure 10 A is shown in by the photo of the sample of GUS activity, root shows that the photo of the sample of medium GUS activity is shown in Figure 10 B, and root shows that the photo of the sample of stronger GUS activity is shown in Figure 10 C, and root does not show that the photo of the sample of GUS activity is shown in Figure 10 D.Figure 10 E is shown in by photo after the GUS dyeing of the root of wheat breed " capital winter No. 1 ", do not show that GUS is active.
2, second group of processing
In step (4), by substratum first, replace substratum second, other Complete Synchronization rapid 1.Cultivate the photo of 3-4 during week and see Fig. 9 B.Many bud points allow to form, and can not normal development become seedling;
Obtain altogether the plant that 101 strains survive, seedling rate is 21%.
In the plant that 101 strains survive, 27 strains are that GUS identifies positive plant, and genetic transformation rate is 26.7%.
3, the 3rd group of processing
In step (4), with embryo callus subculture division culture medium, replace substratum second, other Complete Synchronization rapid 1.
Obtain altogether the plant that 89 strains survive, seedling rate is 51%.
In the plant that 89 strains survive, 15 strains are that GUS identifies positive plant, and genetic transformation rate is 16.9%.
The result of comprehensive step 1 to 3.In step (4), if do not adopt Totomycin to screen, seedling rate is higher, but genetic transformation rate is very low.Adopt 20mgL
-1during hygromycin selection, than adopting 40mgL
-1the genetic transformation rate of hygromycin selection slightly reduces, but seedling rate raises greatly, and the transformation seedlings sum of acquisition has increased greatly, reduces screening and presses the growth that is beneficial to bud point, the normal development rate of raising bud point.
The genetic transformation of embodiment 4, wheat
With TaCYP-pUN1301 carrier, replace pUN1301 carrier, other is completely with embodiment 3.
In first group of processing, seedling rate is 42%, and genetic transformation rate is 13%.
In second group of processing, seedling rate is 16%, and genetic transformation rate is 14%.
In the 3rd group of processing, seedling rate is 59%, and genetic transformation rate is 6%.
Embodiment 5, substratum produce the impact of bud point on embryo callus
One, the preparation of substratum
Substratum I: be only that with the difference of the MS substratum of embodiment 1 sucrose of quality such as using replaces maltose, every liter adds 0.1g inositol.
The preparation method of substratum II: add 2,4 dichlorophenoxyacetic acid, glutamine and caseinhydrolysate in substratum I, the concentration that makes 2,4 dichlorophenoxyacetic acid is 2mgL
-1, glutamine concentration be 500mgL
-1, caseinhydrolysate concentration be 500mgL
-1.
The preparation method of substratum III: add 2,4 dichlorophenoxyacetic acid, glutamine and caseinhydrolysate in substratum I, the concentration that makes 2,4 dichlorophenoxyacetic acid is 1mgL
-1, glutamine concentration be 500mgL
-1, caseinhydrolysate concentration be 500mgL
-1.
The preparation method of substratum IV: add N.F,USP MANNITOL in substratum II, the concentration that makes N.F,USP MANNITOL is 0.4molL
-1.
The preparation method of substratum V: add Totomycin in substratum II, the concentration that makes Totomycin is 40mgL
-1.
The preparation method of substratum VI substratum: add 6-glycosyl aminopurine and caseinhydrolysate in substratum I, the concentration that makes 6-glycosyl aminopurine is 2mgL
-1, caseinhydrolysate concentration be 500mgL
-1.
Two, the preparation of wheat callus
1, get wheat breed " capital winter No. 1 " and bloom after the immature seed of 2 weeks, peel off bran sheet, being placed in 70%(volume ratio) ethanol aqueous solution soaks 30 seconds, with sterile distilled water, rinse 2 times, then be placed in and proceed to the 0.1g/100mL mercuric chloride aqueous solution and soak 10 minutes (sterilization), with sterile distilled water, rinse after 4-5 time, then operation surgical forceps is chosen embryo, shield towards on be placed in substratum II, 25 ℃ within dark culturing 4-5 month, (every 2 weeks subcultures once, during subculture, all adopt substratum III), obtain less, fine and close frangible, the pale yellow vivid callus of particle.
That the callus 2, obtaining from step 1, gets 2.5 centimetres of diameters is placed in substratum IV central authorities, and each culture dish is put 100 left and right callus, 25 ℃ of dark culturing 4 hours.
Three, Wheat Transformation Efficiency By Particle Bombardment callus
Step 2 with embodiment 3.
Four, transform the gradient screening of rear wheat callus
1, first group of processing
(1) callus of completing steps three is placed in substratum IV to 25 ℃ of dark culturing 12 hours.
(2) callus of completing steps (1) is placed in substratum V, 25 ℃ of dark culturing 45 days, the substratum V the subculture that within every 15 days, more renew, the pale yellow callus vivid and molecular marker for increased proliferation of color is resistant calli, and color becomes dun and the callus of not breeding is non-resistance callus.
(3) resistant calli step (2) being obtained is placed in and contains 40mgL
-1in the substratum VI of Totomycin, cultivate 15 days, obtain producing the embryo callus of bud point.Culture condition: 25 ℃, light dark period is 12 hours/12 hours.
Figure 11 is shown in by photo.Compare the bud point comparatively small amt that Figure 11 produces with the Fig. 4 in embodiment 3.
The genetic transformation of embodiment 6, wheat
One, the preparation of substratum
1, the preparation of MS substratum
Step 1 with embodiment 1.
With the MS substratum of step 1 preparation as basic medium, other substratum in preparation steps 2.
2, the preparation of other substratum
The preparation method of embryo callus subculture inducing culture: add 2,4 dichlorophenoxyacetic acid, glutamine and caseinhydrolysate in MS substratum, the concentration that makes 2,4 dichlorophenoxyacetic acid is 1.5mgL
-1, glutamine concentration be 300mgL
-1, caseinhydrolysate concentration be 300mgL
-1.
The preparation method of embryo callus subculture proliferated culture medium: add 2,4 dichlorophenoxyacetic acid, glutamine and caseinhydrolysate in MS substratum, the concentration that makes 2,4 dichlorophenoxyacetic acid is 1.5mgL
-1, glutamine concentration be 300mgL
-1, caseinhydrolysate concentration be 300mgL
-1.
Height oozes the preparation method of substratum: in embryo callus subculture inducing culture, add N.F,USP MANNITOL, the concentration that makes N.F,USP MANNITOL is 0.3molL
-1.
The preparation method of screening culture medium: add Totomycin in embryo callus subculture inducing culture, the concentration that makes Totomycin is 37mgL
-1.
The preparation method of embryo callus subculture division culture medium: add 6-glycosyl aminopurine and caseinhydrolysate in MS substratum, the concentration that makes 6-glycosyl aminopurine is 1.5mgL
-1, caseinhydrolysate concentration be 300mgL
-1.
The preparation method of root media adds α-naphthaleneacetic acid (being commonly called as growth hormone NAA) in MS substratum, and the concentration that makes α-naphthaleneacetic acid is 0.5mgL
-1.
Two, the preparation of wheat callus
1, with Wheat Tissue, replace
The immature seed of 2 weeks after getting wheat breed " spend No. 9 in capital " and blooming, peel off bran sheet, being placed in 70%(volume ratio) ethanol aqueous solution soaks 30 seconds, with sterile distilled water, rinse 2 times, then be placed in and proceed to the 0.1g/100mL mercuric chloride aqueous solution and soak 10 minutes (sterilization), with sterile distilled water, rinse after 4-5 time, then operation surgical forceps is chosen embryo, shield towards on be placed on embryo callus subculture inducing culture, 25 ℃ within dark culturing 4-5 month, (every 2 weeks subcultures once, during subculture, all adopt embryo callus subculture proliferated culture medium), obtain particle less, fine and close frangible, pale yellow vivid callus.
2, the height that is placed in of getting 2.5 centimetres of diameters the callus obtaining from step 1 oozes substratum central authorities, and each culture dish is put 100 left and right callus, 25 ℃ of dark culturing 4 hours.
Three, Wheat Transformation Efficiency By Particle Bombardment callus
The callus that TaCYP-pUN1301 carrier is obtained by particle gun implant steps two.Bombarding conditions: the concentration of TaCYP-pUN1301 carrier is 1 μ g/ μ l; Adopting diameter is the bronze of 1.0 μ m, the split film of 1100psi; Target material to the distance that can split film is 9 centimetres.
Four, transform the gradient screening of rear wheat callus
(1) callus of completing steps three is placed in to height and oozes on substratum, 25 ℃ of dark culturing 12 hours.
(2) callus of completing steps (1) is placed in screening culture medium, 25 ℃ of dark culturing 35 days, the screening culture medium that every 10-15 days more renews subculture, the pale yellow callus vivid and molecular marker for increased proliferation of color is resistant calli, and color becomes dun and the callus of not breeding is non-resistance callus.
(3) resistant calli step (2) being obtained is placed in containing 37mgL
-1on the embryo callus subculture division culture medium of Totomycin, cultivate 10 days, obtain producing the embryo callus of bud point.Culture condition: 25 ℃, light dark period is 12 hours/12 hours.
(4) embryo callus of generation bud point step (3) being obtained is placed in containing 17mgL
-1upper on the embryo callus subculture division culture medium of Totomycin, cultivate 30 days, the identical substratum the subculture that within every 15 days, more renew, bud point develops into seedling.Culture condition: 25 ℃, light dark period is 12 hours/12 hours.
(5) seedling of 2 centimetres of left and right that step (4) obtained is placed on root media, and being cultured to little seedling rooting and root length is 10 centimetres of left and right, opens container closure film, hardening 2-3 days.Culture condition is: 25 ℃, light dark period is 12 hours/12 hours.
Obtain altogether the plant that 105 strains survive.Seedling rate is 47%.
(6) plantlet of transplant step (5) being obtained Nutrition Soil and the vermiculite of mass mixing (be equipped with etc.) in flowerpot is cultivated in phytotron.Culture condition is: 21 ℃ ± 5 ℃, light dark period is 16 hours/8 hours.
(7) by GUS, dye and identify genetic transformation efficiency
Get the root of cultivating the plant of 1 month in step (6), in GUS dye liquor, in 25 ℃ of soaked overnight, show that blue plant has GUS activity, positive plant.
In the plant surviving, the positive plant of 11 strain, genetic transformation rate is 10%.
The genetic transformation of embodiment 7, wheat
One, the preparation of substratum
1, the preparation of MS substratum
Step 1 with embodiment 1.
With the MS substratum of step 1 preparation as basic medium, other substratum in preparation steps 2.
2, the preparation of other substratum
The preparation method of embryo callus subculture inducing culture: add 2,4 dichlorophenoxyacetic acid, glutamine and caseinhydrolysate in MS substratum, the concentration that makes 2,4 dichlorophenoxyacetic acid is 1.5mgL
-1, glutamine concentration be 700mgL
-1, caseinhydrolysate concentration be 700mgL
-1.
The preparation method of embryo callus subculture proliferated culture medium: add 2,4 dichlorophenoxyacetic acid, glutamine and caseinhydrolysate in MS substratum, the concentration that makes 2,4 dichlorophenoxyacetic acid is 1mgL
-1, glutamine concentration be 700mgL
-1, caseinhydrolysate concentration be 700mgL
-1.
Height oozes the preparation method of substratum: in embryo callus subculture inducing culture, add N.F,USP MANNITOL, the concentration that makes N.F,USP MANNITOL is 0.8molL
-1.
The preparation method of screening culture medium: add Totomycin in embryo callus subculture inducing culture, the concentration that makes Totomycin is 43mgL
-1.
The preparation method of embryo callus subculture division culture medium: add 6-glycosyl aminopurine and caseinhydrolysate in MS substratum, the concentration that makes 6-glycosyl aminopurine is 3mgL
-1, caseinhydrolysate concentration be 700mgL
-1.
The preparation method of root media adds α-naphthaleneacetic acid (being commonly called as growth hormone NAA) in MS substratum, and the concentration that makes α-naphthaleneacetic acid is 0.5mgL
-1.
Two, the preparation of wheat callus
1, with Wheat Tissue, replace
The immature seed of 2 weeks after getting wheat breed " spend No. 9 in capital " and blooming, peel off bran sheet, being placed in 70%(volume ratio) ethanol aqueous solution soaks 30 seconds, with sterile distilled water, rinse 2 times, then be placed in and proceed to the 0.1g/100mL mercuric chloride aqueous solution and soak 10 minutes (sterilization), with sterile distilled water, rinse after 4-5 time, then operation surgical forceps is chosen embryo, shield towards on be placed on embryo callus subculture inducing culture, 25 ℃ within dark culturing 4-5 month, (every 2 weeks subcultures once, during subculture, all adopt embryo callus subculture proliferated culture medium), obtain particle less, fine and close frangible, pale yellow vivid callus.
2, the height that is placed in of getting 2.5 centimetres of diameters the callus obtaining from step 1 oozes substratum central authorities, and each culture dish is put 100 left and right callus, 25 ℃ of dark culturing 4 hours.
Three, Wheat Transformation Efficiency By Particle Bombardment callus
The callus that TaCYP-pUN1301 carrier is obtained by particle gun implant steps two.Bombarding conditions: the concentration of TaCYP-pUN1301 carrier is 1 μ g/ μ l; Adopting diameter is the bronze of 1.0 μ m, the split film of 1100psi; Target material to the distance that can split film is 9 centimetres.
Four, transform the gradient screening of rear wheat callus
(1) callus of completing steps three is placed in to height and oozes on substratum, 25 ℃ of dark culturing 12 hours.
(2) callus of completing steps (1) is placed in screening culture medium, 25 ℃ of dark culturing 55 days, the screening culture medium that every 10-15 days more renews subculture, the pale yellow callus vivid and molecular marker for increased proliferation of color is resistant calli, and color becomes dun and the callus of not breeding is non-resistance callus.
(3) resistant calli step (2) being obtained is placed in containing 43mgL
-1on the embryo callus subculture division culture medium of Totomycin, cultivate 20 days, obtain producing the embryo callus of bud point.Culture condition: 25 ℃, light dark period is 12 hours/12 hours.
(4) embryo callus of generation bud point step (3) being obtained is placed in containing 23mgL
-1upper on the embryo callus subculture division culture medium of Totomycin, cultivate 60 days, the identical substratum the subculture that within every 15 days, more renew, bud point develops into seedling.Culture condition: 25 ℃, light dark period is 12 hours/12 hours.
(5) seedling of 2 centimetres of left and right that step (4) obtained is placed on root media, and being cultured to little seedling rooting and root length is 10 centimetres of left and right, opens container closure film, hardening 2-3 days.Culture condition is: 25 ℃, light dark period is 12 hours/12 hours.
Obtain altogether the plant that 86 strains survive.Seedling rate is 37%.
(6) plantlet of transplant step (5) being obtained Nutrition Soil and the vermiculite of mass mixing (be equipped with etc.) in flowerpot is cultivated in phytotron.Culture condition is: 21 ℃ ± 5 ℃, light dark period is 16 hours/8 hours.
(7) by GUS, dye and identify genetic transformation efficiency
Get the root of cultivating the plant of 1 month in step (6), in GUS dye liquor, in 25 ℃ of soaked overnight, show that blue plant has GUS activity, positive plant.
In the plant surviving, the positive plant of 13 strain, genetic transformation rate is 15%.
Claims (6)
1. an Efficiency of Wheat Transformation method, comprises the steps:
(1) wheat callus is carried out to preculture;
(2) expression vector that contains goal gene is imported to the embryo callus of completing steps (1) by particle bombardment; Described expression vector contains hygromycin gene;
(3) embryo callus of completing steps (2) is carried out to renewal cultivation;
(4) get the embryo callus of completing steps (3), in screening culture medium, cultivate 35-55 days; In described screening culture medium, contain 35-45mgL
-1totomycin;
(5) get the embryo callus that step (4) obtains, containing 35-45mgL
-1on the division culture medium of Totomycin, cultivate 10-20 days, obtain producing the embryo callus of bud point;
(6) get the embryo callus of the generation bud point that step (5) obtains, containing 15-25mgL
-1on the division culture medium of Totomycin, cultivate 30-60 days, obtain seedling;
(7) get the seedling that step (6) obtains, on root media, be cultured to and take root.
2. the method for claim 1, is characterized in that: in described step (1), the substratum that described preculture adopts is that height oozes substratum.
3. method as claimed in claim 1 or 2, is characterized in that: in described step (3), the substratum that described renewal cultivation adopts is that height oozes substratum.
4. the method for claim 1, is characterized in that: in described step (4), described screening culture medium is the substratum that adds Totomycin to obtain in embryo callus subculture inducing culture.
5. the method for claim 1, is characterized in that: in described step (5) and/or described step (6), described division culture medium is embryo callus subculture division culture medium.
6. the application of arbitrary described method in wheat breeding in claim 1 to 5.
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