CN106148398A - A kind of culture medium reducing wheat anther white Seedling rate and conversion method for agrobacterium thereof - Google Patents
A kind of culture medium reducing wheat anther white Seedling rate and conversion method for agrobacterium thereof Download PDFInfo
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Abstract
The present invention " a kind of culture medium reducing wheat anther white Seedling rate and conversion method for agrobacterium thereof ", belongs to genetic transfoumation, and described MC17 culture medium is to the addition of PAA and colchicine on the basis of C17, instead of sucrose with maltose.Present invention request additionally provides genetic transforming method based on this culture medium, it is possible to significantly improves callus induction rate, resistant plant pick-up rate, reduces white Seedling rate, can actively promote wheat transgenic research.
Description
Technical field
The present invention relates to genetic transfoumation, a kind of culture medium reducing wheat anther white Seedling rate and conversion method for agrobacterium.
Background technology
Agrobacterium-mediated genetic transformation method is the common method of current plant genetic engineering, compares with methods such as via Particle Bombardment Transformations,
Agrobacterium-mediated transformation has simple, it is not necessary to expensive instrument and equipment, and transgenic is many is integrated into receptor dyeing with single low copy
Body, the feature such as the heredity of transgenic plant offspring is relatively stable, the method is constantly subjected to universal in terms of plant transgene research
Pay attention to and application.Up to now, the transgenic plant that medium agrobacterium co-cultivation obtains accounts for about the 85% of transgenic plant sum.With
Time, some have the exogenous gene of value import plant, and gradually transgenic plant is applied in agricultural production, turn base
Because the New Crop Varieties such as Semen sojae atricolor, Semen Maydis, Cotton Gossypii, Brassica campestris L industrialization area on producing increases year by year.
In cereal crops, Semen Tritici aestivi belongs to the crop of genetic transformation difficulty the most, adds that transgenic research is started late, gene work
Journey breeding process lags significantly behind other crops.From Vasil in 1992 etc., GUS/Bar channel genes Semen Tritici aestivi is obtained the first and turn base
Since Semen Tritici aestivi, transgenic wheat research has had incremental advances, and agrobacterium-mediated transformation is considered as the one of Efficiency of Wheat Transformation always
Road difficulty, but Cheng is equal within 1997, utilizing agrobacterium-mediated transformation to obtain normal transgenic wheat, subsequently state first
Inside and outside other several laboratorys such as Xia etc., leaf are made the country prosperous etc., Wang Yongqin etc., Khanna etc., summer are photosensitive etc. reports the most in succession
Agriculture bacillus mediated Efficiency of Wheat Transformation.
So far, although the research to agrobacterium co-cultivation mediated wheat genetic transformation has a lot, mostly with wheat immature embryo, mature embryo
For acceptor material, it is an exogenous gene heterozygote with rataria, mature embryo for the transgenic line that acceptor material obtains, must be through more certainly
Handing over for 2 generations selected to obtain the individuality that exogenous gene isozygotys, the procedure of breeding is complicated.And carry out heredity with flower pesticide for acceptor material and turn
Changing, not only callus regeneration special type is good, and regrowth just can become, after directly doubling, the stable amphiploid that genotype is isozygotied.
Although agriculture bacillus mediated wheat anther genetic transformation has the advantage of its uniqueness, but the albinism of regeneration plant is extremely widespread,
Bai Miao leads and is typically about 40%, and high reaches 80-90%, the most all white Seedlings so that agriculture bacillus mediated wheat anther can not
Obtain sufficient amount of regeneration plant, the actual requirement relying on this genetic transforming method improvement Semen Tritici aestivi can not be met far away, limit
The large-scale production of transgenic wheat and the development of Semen Tritici aestivi functional genome research.Therefore, Optimization of Wheat induction of anther callus
Culture medium, reduces white Seedling rate and the preliminary conversion method for agrobacterium set up, and huge promotion will will be had to make wheat transgenic research
With.
Summary of the invention
Blank based on above-mentioned field and demand, it is an object of the invention to provide a kind of culture medium reducing wheat anther white Seedling rate and
Wheat genetic transformation method.Technical scheme is as follows:
A kind of culture medium reducing wheat anther white Seedling rate, it is characterised in that formula is as follows: anhydrous calcium chloride 150mg/L, nitric acid
Potassium 1400mg/L, Magnesium sulfate heptahydrate 150mg/L, ammonium nitrate 300mg/L, potassium dihydrogen phosphate 400mg/L；Manganese sulfate 8.5mg/L,
Zinc sulfate 8.6mg/L, boric acid 6.2mg/L, potassium iodide 0.83mg/L, copper sulphate pentahydrate 0.025mg/L, cobalt chloride hexahydrate
0.025mg/L；Ferrous sulfate 27.85mg/L, sodium ethylene diamine tetracetate 37.25mg/L；Glycine 1mg/L, VB1 0.5mg/L,
VB6 0.25mg/L, nicotinic acid 0.25mg/L, biotin 1mg/L, leucine 1mg/L, 2,4-D 2mg/L, KT 0.5mg/L,
Lactoalbumin hydrolysate 500mg/L, PAA 0.5-10mg/L, colchicine 0.005-0.1mg/L, maltose 30-100g/L, agar
3.7g/L。
In above-mentioned culture medium, the concentration of PAA is preferably 1mg/L, the concentration of colchicine is preferably 0.01mg/L, maltose
Concentration is preferably 90g/L.
A kind of agriculture bacillus mediated wheat anther genetic transforming method, comprises the steps:
(1) inducing culture: obtain wheat anther callus,
(2) infect: use the Agrobacterium carrying target gene to infect the wheat anther callus that step (1) obtains,
It is characterized in that: the culture medium that described inducing culture uses is the culture medium described in claim 1 or 2.
The condition of described inducing culture is: cultivation temperature is 27～30 DEG C, cultivates 40-60 days under dark condition.
The wheat anther that described inducing culture uses picks up from and is developed in monokaryon-the wheat children tassel in late period, disappears with the calcium hypochlorite of 10%
Poison 15min, then rinses with sterile distilled water.
The described culture fluid formula that infects used of contaminating is: potassium nitrate 190mg/L, ammonium nitrate 165mg/L, potassium dihydrogen phosphate
17mg/L, anhydrous calcium chloride 44mg/L, Magnesium sulfate heptahydrate 37mg/L；Potassium iodide 0.083mg/L, four water manganese sulfate 2.23mg/L,
Boric acid 0.62mg/L, zinc sulphate heptahydrate 0.86mg/L, cobalt chloride hexahydrate 0.0025mg/L, copper sulphate pentahydrate 0.0025mg/L,
Sodium Molybdate Dihydrate 0.025mg/L；Ferrous sulfate 2.785mg/L, sodium ethylene diamine tetracetate 3.725mg/L；Glycine 2mg/L,
VB1 0.4mg/L, VB6 0.5mg/L, nicotinic acid 0.5mg/L, inositol 100mg/L；Maltose 40g/L, ethyl sulfonic acid (MES) 1.95g/L,
Glutamine 0.1g/L, caseinhydrolysate 0.1g/L, glucose 10g/L, vitamin C 100mg/L, 4-amino-3,5,6-tri-
Chloropyridine formic acid (Picloram) 1mg/L, acetosyringone (AS) 39mg/L, pluronic F68 1ml/L.
Described infect after, also include co-culturing, resistant calli recovery, differentiation culture, root culture step；Resistance is more
Injured tissue recover use culture medium as follows: potassium nitrate 1900mg/L, ammonium nitrate 1650mg/L, potassium dihydrogen phosphate 170mg/L,
Anhydrous calcium chloride 440mg/L, Magnesium sulfate heptahydrate 370mg/L；Potassium iodide 0.83mg/L, four water manganese sulfate 22.3mg/L, boric acid
6.2mg/L, zinc sulphate heptahydrate 8.6mg/L, cobalt chloride hexahydrate 0.025mg/L, copper sulphate pentahydrate 0.025mg/L, Sodium Molybdate Dihydrate
0.25mg/L；Ferrous sulfate 27.85mg/L, sodium ethylene diamine tetracetate 37.25mg/L；Glycine 2mg/L, VB1 0.4mg/L,
VB6 0.5mg/L, nicotinic acid 0.5mg/L, inositol 100mg/L；Sucrose 30g/L, Phytagel 2.7g/L, Dicamba 2mg/L,
Carbenicillin 250mg/L, PPT 3mg/L；
Condition of culture is: cultivate 15-20 days under 25 DEG C of dark conditions.
The culture medium prescription that described differentiation culture uses is: potassium nitrate 1900/L mg, ammonium nitrate 1650mg/L, potassium dihydrogen phosphate
170mg/L, anhydrous calcium chloride 440mg/L, Magnesium sulfate heptahydrate 370mg/L；Potassium iodide 0.83mg/L, four water manganese sulfate 22.3mg/L,
Boric acid 6.2mg/L, zinc sulphate heptahydrate 8.6mg/L, cobalt chloride hexahydrate 0.025mg/L, copper sulphate pentahydrate 0.025mg/L, two water
Sodium molybdate 0.25mg/L；Ferrous sulfate 27.85mg/L, sodium ethylene diamine tetracetate 37.25mg/L；Glycine 2mg/L, VB1
0.4mg/L, VB6 0.5mg/L, nicotinic acid 0.5mg/L, inositol 100mg/L；Vitamin C 100mg/L, 2,4-D 0.5mg/L,
Sucrose 30g/L, Phytagel 2.7g/L, Carbenicillin 250mg/L, PPT 3mg/L；
Condition of culture is: under the conditions of 25 DEG C, and illumination every day carries out differentiation culture in 12-14 hour.
The prescription of rooting medium that described root culture uses is: potassium nitrate 950/L mg, ammonium nitrate 825mg/L, biphosphate
Potassium 85mg/L, anhydrous calcium chloride 220mg/L, Magnesium sulfate heptahydrate 185mg/L；Potassium iodide 0.83mg/L, four water manganese sulfates
22.3mg/L, boric acid 6.2mg/L, zinc sulphate heptahydrate 8.6mg/L, cobalt chloride hexahydrate 0.025mg/L, copper sulphate pentahydrate 0.025mg/L,
Sodium Molybdate Dihydrate 0.25mg/L；Ferrous sulfate 27.85mg/L, sodium ethylene diamine tetracetate 37.25mg/L；Glycine 2mg/L, VB1
0.4mg/L, VB6 0.5mg/L, nicotinic acid 0.5mg/L, inositol 100mg/L；Naphthalene acetic acid (NAA) 1mg/L, paclobutrazol 3mg/L,
Sucrose 80g/L, Phytagel 2.7g/L, Carbenicillin 250mg/L；
Condition of culture is: when the resistance regeneration bud length that callus differentiates is to about 2-3cm, proceeded to described root media
On carry out strong root and cultivate, when adventitious root grows to 3-4cm, test tube Seedling is transferred in 2-4 DEG C of refrigerator carry out vernalization treatment.
The present invention is by improving the callus inducing medium for wheat anther, so that callus induction rate
Being improved with the pick-up rate of resistant plant, Bai Miao leads reduction.Test data shows, in the duplicate feelings of other condition of culture
Under condition, using the callus inducing medium of the present invention, the callus induction rate of Semen Tritici aestivi stone 4185 is 83.4%, Semen Tritici aestivi Ji
The callus induction rate of 5265 is 78.2%；The average pick-up rate of Semen Tritici aestivi stone 4185 resistant plant is 39.0%, Semen Tritici aestivi Ji 5265
The average pick-up rate of resistant plant is 36.3%；The white Seedling rate of Semen Tritici aestivi stone 4185 is 25.4%, and the white Seedling rate of Semen Tritici aestivi Ji 5265 is
23.5%.And using the C17 used in prior art is callus inducing medium, the callus of Semen Tritici aestivi Ji 5265 lures
Conductance is 49.7%, and resistant plant pick-up rate is 20.3%, and it is 46.5% that Bai Miao leads.Experimental data illustrates, in the present invention, and improvement
Callus inducing medium improving callus induction rate and the pick-up rate of resistant plant and play certainly in terms of reducing white Seedling rate
Qualitative effect, therefore, the present invention is claimed the callus inducing medium formula of improvement.
Callus inducing medium based on above-mentioned improvement, the present invention is claimed agriculture bacillus mediated wheat anther further and loses
Pass method for transformation.The method except use culture medium be above-mentioned improvement callus inducing medium in addition to, other step and generation
It can be the step of agriculture bacillus mediated wheat anther genetic transforming method conventional in the prior art that uses of those skilled in the art
And parameter.These steps listed in the embodiment of the present invention and a kind of preferred implementation of parameter, not having the present invention
Restriction effect.
In the present invention, callus induction rate refers to that callus number accounts for the percentage rate of inoculation flower pesticide number, averagely obtaining of resistant plant
Yield refers to that resistance Seedling accounts for the percentage rate of callus number, and Bai Miao leads the percentage rate referring to that Bai Miao accounts for callus number.
Accompanying drawing explanation
Fig. 1 is to use the wheat anther callus inducing culture in existing document that the flower pesticide of Semen Tritici aestivi Ji 5265 carries out induction training
The effect supported.
Fig. 2 is that the wheat anther callus inducing culture using the present invention carries out inducing culture to the flower pesticide of Semen Tritici aestivi Ji 5265
Effect.
Fig. 3 is culture medium of the present invention and method for transformation carries out the induction after Agrobacterium is infected to the anther callus of Semen Tritici aestivi Ji 5265
The culture effect of resistant calli.
Fig. 4 is the differentiation situation of the resistant calli behind culture medium of the present invention and method for transformation transformed wheat Ji 5265.
Fig. 5 is root culture situation behind culture medium of the present invention and method for transformation transformed wheat Ji 5265.
Detailed description of the invention
Experimental technique involved in following embodiment is conventional method if no special instructions.
Embodiment 1, Agrobacterium-mediated Transformation method transformed wheat flower pesticide
One, the preparation of culture medium and sterilizing
1.MC17 culture medium, formula is that every 1000ml contains: anhydrous calcium chloride 150mg, potassium nitrate 1400mg, seven water sulphuric acid
Magnesium 150mg, ammonium nitrate 300mg, potassium dihydrogen phosphate 400mg；Manganese sulfate 8.5mg, zinc sulfate 8.6mg, boric acid 6.2mg,
Potassium iodide 0.83mg, copper sulphate pentahydrate 0.025mg, cobalt chloride hexahydrate 0.025mg；Ferrous sulfate 27.85mg, ethylenediamine tetrem
Acid sodium 37.25mg；Glycine 1mg, VB1 0.5mg, VB6 0.25mg, nicotinic acid 0.25mg, biotin 1mg, leucine
1mg, 2,4-D 2mg, KT 0.5mg, PAA 1mg, colchicine 0.01mg, lactoalbumin hydrolysate 500mg, maltose
90g, agar 3.7g.
Compound method is as follows: anhydrous calcium chloride 150mg, potassium nitrate 1400mg, Magnesium sulfate heptahydrate 150mg, ammonium nitrate 300mg,
Potassium dihydrogen phosphate 400mg；Manganese sulfate 8.5mg, zinc sulfate 8.6mg, boric acid 6.2mg, potassium iodide 0.83mg, copper sulphate pentahydrate
0.025mg, cobalt chloride hexahydrate 0.025mg；Ferrous sulfate 27.85mg, sodium ethylene diamine tetracetate 37.25mg；Glycine 1mg,
VB1 0.5mg, VB6 0.25mg, nicotinic acid 0.25mg, biotin 1mg, leucine 1mg, 2,4-D 2mg, KT 0.5mg,
Lactoalbumin hydrolysate 500mg, maltose 90g, constant volume 1000ml, regulate PH to 5.8, add agar 3.7g, above one-tenth
Point use 121 DEG C of autoclavings 20 minutes, subject to sterilization after culture medium temperature be down to add filtration sterilization when about 65 DEG C after
PAA 1mg, colchicine 0.01mg.
The feature of MC17 culture medium is to the addition of PAA and colchicine, instead of sucrose with maltose.
2. infecting culture medium, formula is that every 1000ml contains: potassium nitrate 190mg, ammonium nitrate 165mg, potassium dihydrogen phosphate 17mg,
Anhydrous calcium chloride 44mg, Magnesium sulfate heptahydrate 37mg；Potassium iodide 0.083mg, four water manganese sulfate 2.23mg, boric acid 0.62mg,
Zinc sulphate heptahydrate 0.86mg, cobalt chloride hexahydrate 0.0025mg, copper sulphate pentahydrate 0.0025mg, Sodium Molybdate Dihydrate 0.025mg；Sulfur
Acid ferrous 2.785mg, sodium ethylene diamine tetracetate 3.725mg；Glycine 2mg, VB1 0.4mg, VB6 0.5mg, nicotinic acid 0.5mg,
Inositol 100mg；Maltose 40g, ethyl sulfonic acid (MES) 1.95g, glutamine 0.1g, caseinhydrolysate 0.1g, glucose 10g,
Vitamin C 100mg, 4-amino-3,5,6-trichloropicolinic acid (Picloram) 1mg, acetosyringone (AS) 39mg,
pluronic F68 1ml。
Compound method is as follows: potassium nitrate 190mg, ammonium nitrate 165mg, potassium dihydrogen phosphate 17mg, anhydrous calcium chloride 44mg,
Magnesium sulfate heptahydrate 37mg；Potassium iodide 0.083mg, four water manganese sulfate 2.23mg, boric acid 0.62mg, zinc sulphate heptahydrate 0.86mg,
Cobalt chloride hexahydrate 0.0025mg, copper sulphate pentahydrate 0.0025mg, Sodium Molybdate Dihydrate 0.025mg；Ferrous sulfate 2.785mg, second
Sodium ethylene diamine tetracetate 3.725mg；Maltose 40g, ethyl sulfonic acid (MES) 1.95g, constant volume 950ml, regulate PH to 5.4, above
Composition uses 121 DEG C of autoclavings 20 minutes；Uniformly mix following several compositions: 100 × MS vitamin 10ml is (containing VB1
0.4mg, VB6 0.5mg, nicotinic acid 0.5mg, inositol 100mg), glutamine 0.1g, caseinhydrolysate 0.1g, glucose
10g, vitamin C 100mg, 4-amino-3,5,6-trichloropicolinic acid (Picloram) 1mg, acetosyringone (AS)
39mg, pluronic F68 1ml, constant volume 50ml, add after filtration sterilization in the culture medium after above-mentioned autoclaving.
3. resistant calli recovery media, formula is that every 1000ml contains: potassium nitrate 1900mg, ammonium nitrate 1650mg, phosphorus
Acid dihydride potassium 170mg, anhydrous calcium chloride 440mg, Magnesium sulfate heptahydrate 370mg；Potassium iodide 0.83mg, four water manganese sulfate 22.3mg,
Boric acid 6.2mg, zinc sulphate heptahydrate 8.6mg, cobalt chloride hexahydrate 0.025mg, copper sulphate pentahydrate 0.025mg, Sodium Molybdate Dihydrate
0.25mg；Ferrous sulfate 27.85mg, sodium ethylene diamine tetracetate 37.25mg；Glycine 2mg, VB1 0.4mg, VB6 0.5mg,
Nicotinic acid 0.5mg, inositol 100mg；Sucrose 30g, Phytagel 2.7g, Dicamba 2mg, Carbenicillin 250mg, PPT 3mg.
Compound method is as follows: potassium nitrate 1900mg, ammonium nitrate 1650mg, potassium dihydrogen phosphate 170mg, anhydrous calcium chloride 440mg,
Magnesium sulfate heptahydrate 370mg；Potassium iodide 0.83mg, four water manganese sulfate 22.3mg, boric acid 6.2mg, zinc sulphate heptahydrate 8.6mg,
Cobalt chloride hexahydrate 0.025mg, copper sulphate pentahydrate 0.025mg, Sodium Molybdate Dihydrate 0.25mg；Ferrous sulfate 27.85mg, ethylenediamine
Tetraacethyl sodium 37.25mg；Glycine 2mg, VB1 0.4mg, VB6 0.5mg, nicotinic acid 0.5mg, inositol 100mg, sucrose
30g, constant volume 1000ml, regulate PH to 5.8, adds Phytagel 2.7g, and above composition uses 121 DEG C of autoclavings 20 points
Clock, subject to sterilization after culture medium temperature add the Dicamba 2mg after filtration sterilization, Carbenicillin when being down to about 65 DEG C
250mg、PPT 3mg。
4. callus division culture medium, formula is that every 1000ml contains: potassium nitrate 1900mg, ammonium nitrate 1650mg, di(2-ethylhexyl)phosphate
Hydrogen potassium 170mg, anhydrous calcium chloride 440mg, Magnesium sulfate heptahydrate 370mg；Potassium iodide 0.83mg, four water manganese sulfate 22.3mg,
Boric acid 6.2mg, zinc sulphate heptahydrate 8.6mg, cobalt chloride hexahydrate 0.025mg, copper sulphate pentahydrate 0.025mg, Sodium Molybdate Dihydrate
0.25mg；Ferrous sulfate 27.85mg, sodium ethylene diamine tetracetate 37.25mg；Glycine 2mg, VB1 0.4mg, VB6 0.5mg,
Nicotinic acid 0.5mg, inositol 100mg；Vitamin C 100mg, 2,4-D 0.5mg, sucrose 30g, Phytagel 2.7g, carboxylic benzyl
Penicillin 250mg, PPT 3mg.
Compound method is as follows: potassium nitrate 1900mg, ammonium nitrate 1650mg, potassium dihydrogen phosphate 170mg, anhydrous calcium chloride 440mg,
Magnesium sulfate heptahydrate 370mg；Potassium iodide 0.83mg, four water manganese sulfate 22.3mg, boric acid 6.2mg, zinc sulphate heptahydrate 8.6mg,
Cobalt chloride hexahydrate 0.025mg, copper sulphate pentahydrate 0.025mg, Sodium Molybdate Dihydrate 0.25mg；Ferrous sulfate 27.85mg, ethylenediamine
Tetraacethyl sodium 37.25mg；Glycine 2mg, VB1 0.4mg, VB6 0.5mg, nicotinic acid 0.5mg, inositol 100mg, dimension are raw
Element C 100mg, 2,4-D 0.5mg, sucrose 30g, constant volume 1000ml, regulate PH to 5.8, add Phytagel 2.7g, with
Upper composition uses 121 DEG C of autoclavings 20 minutes, subject to sterilization after culture medium temperature be down to add filtration sterilization when about 65 DEG C after
Carbenicillin 250mg, PPT 3mg.
5. root media, formula is that every 1000ml contains: potassium nitrate 950mg, ammonium nitrate 825mg, potassium dihydrogen phosphate 85mg,
Anhydrous calcium chloride 220mg, Magnesium sulfate heptahydrate 185mg；Potassium iodide 0.83mg, four water manganese sulfate 22.3mg, boric acid 6.2mg,
Zinc sulphate heptahydrate 8.6mg, cobalt chloride hexahydrate 0.025mg, copper sulphate pentahydrate 0.025mg, Sodium Molybdate Dihydrate 0.25mg；Sulphuric acid is sub-
Ferrum 27.85mg, sodium ethylene diamine tetracetate 37.25mg；Glycine 2mg, VB1 0.4mg, VB6 0.5mg, nicotinic acid 0.5mg,
Inositol 100mg；Naphthalene acetic acid (NAA) 1mg, paclobutrazol 3mg, sucrose 80g, Phytagel 2.7g, Carbenicillin 250mg.
Compound method is as follows: potassium nitrate 950mg, ammonium nitrate 825mg, potassium dihydrogen phosphate 85mg, anhydrous calcium chloride 220mg,
Magnesium sulfate heptahydrate 185mg；Potassium iodide 0.83mg, four water manganese sulfate 22.3mg, boric acid 6.2mg, zinc sulphate heptahydrate 8.6mg,
Cobalt chloride hexahydrate 0.025mg, copper sulphate pentahydrate 0.025mg, Sodium Molybdate Dihydrate 0.25mg；Ferrous sulfate 27.85mg, ethylenediamine
Tetraacethyl sodium 37.25mg；Glycine 2mg, VB1 0.4mg, VB6 0.5mg, nicotinic acid 0.5mg, inositol 100mg；, naphthalene second
Acid (NAA) 1mg, paclobutrazol 3mg, sucrose 80g, constant volume 1000ml, regulate PH to 5.8, add Phytagel 2.7g,
Above composition uses 121 DEG C of autoclavings 20 minutes, subject to sterilization after culture medium temperature be down to when about 65 DEG C add filtration sterilization
After Carbenicillin 250mg.
Two, Agrobacterium-mediated Transformation method transformed wheat flower pesticide
1, inducing culture
The former microscopy of drawing materials of late April Semen Tritici aestivi heading.Choose Wheat Pollen cell development to the wheat children tassel of monokaryon middle and advanced stage, use
The calcium hypochlorite sterilization 15min of 10%, then rinses 3 times with sterile distilled water, each 3min.Super-clean bench is inoculated flower pesticide
In MC17 culture medium, light culture inducing wheat anther callus under the conditions of 28 DEG C.
The flower pesticide of Semen Tritici aestivi Ji 5265 carries out the effect of inducing culture as shown in Figure 2.The callus induction rate of Semen Tritici aestivi stone 4185 is
83.4% (callus induction rate refers to that callus number accounts for the percentage rate of inoculation flower pesticide number), the callus of Semen Tritici aestivi Ji 5265 lures
Conductance is 78.2%.
2, infect
By in single colony inoculation of purpose Agrobacterium to YEP culture fluid, under 28 DEG C of dark conditions, 250rpm concussion is cultivated to OD
Value is between 0.6-1.0.Then OD value Agrobacterium 4500rpm under room temperature condition between 0.6-1.0 is centrifuged 10min,
Collection precipitation is resuspended in and infects in culture fluid.The callus of wheat anther is put into during resuspended bacterium solution is soaked 30min. light
Gently rock.
3, co-culture
Take out the anther callus after purpose Agrobacterium is infected, be placed on aseptic filter paper (infecting liquid a little), 25 DEG C of dark bars
2 days are co-cultured under part.
4, induction of resistance callus
Wheat anther callus after co-culturing is placed on resistant calli inducing culture, cultivates under 25 DEG C of dark conditions
15-20 days induction of resistance calluss.
The anther callus of Semen Tritici aestivi Ji 5265 carries out culture effect such as Fig. 3 institute of the induction of resistance callus after Agrobacterium is infected
Show.
5, callus differentiation
The resistant calli of wheat anther is transferred on callus division culture medium, under the conditions of 25 DEG C, illumination every day 12-14
Hour carry out differentiation culture.The differentiation situation of the resistant calli behind Semen Tritici aestivi Ji 5265 is as shown in Figure 4.
6, root culture
From the resistance regeneration bud length that wheat anther resistant calli differentiates to about 2-3cm time, proceeded to root media
On, carry out strong root and cultivate, when adventitious root grows to 3-4cm, test tube Seedling is transferred in 2-4 DEG C of refrigerator carry out vernalization treatment.Little
Behind wheat Ji 5265, root culture situation is as shown in Figure 5.
7, chromosome doubling
Plant good for vernalization is taken out, thoroughly removes root culture medium, be transferred in soil cultivate, Wheat Pollen is transplanted
Survival and growth, to when dividing evil to contain the phase, differentiates Ploidy of Wheat Pollen Plants, guard cell by Guard Cell Length under the microscope
It is considered amphiploid without doubling, the colchicine of the plant 0.75-1% of below 65u and 1.5% dimethyl at more than 65u
Sulfoxide mixed liquor soaks tillering node 4-5 hour under the conditions of 25 DEG C, and the dyed body of pollen plant obtains after doubling doubling isozygotying
Body.
8, Molecular Detection
Carry out Molecular Detection with conventional molecular detection technology to doubling homozygous individual, screen stable transgenic homozygous strain.
Result of the test: the average pick-up rate of Semen Tritici aestivi stone 4185 resistant plant is 39.0%, Semen Tritici aestivi Ji 5265 resistant plant average
Pick-up rate is 36.3%；The white Seedling rate of Semen Tritici aestivi stone 4185 is 25.4%, and the white Seedling rate of Semen Tritici aestivi Ji 5265 is 23.5%.
Embodiment 2
The present embodiment is identical with the step of embodiment 1, and difference is the step 1 of the present embodiment) inducing wheat anther callus
Dark culturing temperature be 30 DEG C.
The callus induction rate of Semen Tritici aestivi Ji 5265 is 87.4%, and resistant plant pick-up rate is 25.1%, and it is 35.2% that Bai Miao leads.
Embodiment 3
The present embodiment is identical with the step of embodiment 1, and difference is the step 1 of the present embodiment) inducing wheat anther callus
Culture medium be sent the documents the conventional inducing culture C17 in offering (Wang Pei, Chen Yurong, improve Winter Wheat Anther Plantlets inductivity
Research, North China agronomy report, 1986,1 (1): 20-25).The callus induction rate of Semen Tritici aestivi Ji 5265 is 49.7%, anti-
Property plant pick-up rate is 20.3%, and it is 46.5% that Bai Miao leads.
The present invention uses PAA to take 0.5-2mg/L, colchicine 0.005-0.05mg/L, the MC17 of maltose 30-100g/L make
Carry out similar embodiment 1, the test of 2,3 for callus inducing medium, draw essentially identical result, the i.e. present invention
Callus inducing medium in the range of being claimed, relative to conventional inducing culture C17, has raising callus induction
Rate, resistant plant pick-up rate, and reduce the effect of white Seedling rate.
Claims (10)
1. the culture medium reducing wheat anther white Seedling rate, it is characterised in that formula is as follows: anhydrous calcium chloride 150mg/L, nitre
Acid potassium 1400mg/L, Magnesium sulfate heptahydrate 150mg/L, ammonium nitrate 300mg/L, potassium dihydrogen phosphate 400mg/L；Manganese sulfate 8.5mg/L,
Zinc sulfate 8.6mg/L, boric acid 6.2mg/L, potassium iodide 0.83mg/L, copper sulphate pentahydrate 0.025mg/L, cobalt chloride hexahydrate
0.025mg/L；Ferrous sulfate 27.85mg/L, sodium ethylene diamine tetracetate 37.25mg/L；Glycine 1mg/L, VB1 0.5mg/L,
VB6 0.25mg/L, nicotinic acid 0.25mg/L, biotin 1mg/L, leucine 1mg/L, 2,4-D 2mg/L, KT 0.5mg/L,
Lactoalbumin hydrolysate 500mg/L, PAA 0.5-10mg/L, colchicine 0.005-0.1mg/L, maltose 30-100g/L, agar
3.7g/L。
The culture medium of reduction wheat anther the most according to claim 1 white Seedling rate, it is characterised in that: the concentration of PAA is
1mg/L, the concentration of colchicine are 0.01mg/L, the concentration of maltose is 90g/L.
3. an agriculture bacillus mediated wheat anther genetic transforming method, comprises the steps:
(1) inducing culture: obtain wheat anther callus,
(2) infect: use the Agrobacterium carrying target gene to infect the wheat anther callus that step (1) obtains,
It is characterized in that: the culture medium that described inducing culture uses is the culture medium described in claim 1 or 2.
Wheat anther genetic transforming method the most according to claim 3, goes to be characterised by, the condition of described inducing culture is:
Cultivation temperature is 27～30 DEG C, cultivates 40-60 days under dark condition.
Wheat anther genetic transforming method the most according to claim 3, goes to be characterised by, it is little that described inducing culture uses
Wheat flower pesticide picks up from and is developed in monokaryon-the wheat children tassel in late period, and the calcium hypochlorite with 10% is sterilized 15min, then uses aseptic distillation
Water rinses.
Wheat anther genetic transforming method the most according to claim 3, goes to be characterised by, what described dip-dye used infects training
Nutrient solution formula is: potassium nitrate 190mg/L, ammonium nitrate 165mg/L, potassium dihydrogen phosphate 17mg/L, anhydrous calcium chloride 44mg/L,
Magnesium sulfate heptahydrate 37mg/L；Potassium iodide 0.083mg/L, four water manganese sulfate 2.23mg/L, boric acid 0.62mg/L, zinc sulphate heptahydrate
0.86mg/L, cobalt chloride hexahydrate 0.0025mg/L, copper sulphate pentahydrate 0.0025mg/L, Sodium Molybdate Dihydrate 0.025mg/L；Sulphuric acid
Ferrous 2.785mg/L, sodium ethylene diamine tetracetate 3.725mg/L；Glycine 2mg/L, VB1 0.4mg/L, VB6 0.5mg/L,
Nicotinic acid 0.5mg/L, inositol 100mg/L；Maltose 40g/L, ethyl sulfonic acid (MES) 1.95g/L, glutamine 0.1g/L, hydrolysis
Casein 0.1g/L, glucose 10g/L, vitamin C 100mg/L, 4-amino-3,5,6-trichloropicolinic acid (Picloram)
1mg/L, acetosyringone (AS) 39mg/L, pluronic F68 1ml/L.
7., according to the arbitrary described wheat anther genetic transforming method of claim 3～6, go to be characterised by, described in infect after,
Also include co-culturing, induction of resistance callus, differentiation culture, root culture, chromosome doubling step；
Described resistant calli recover use culture medium prescription as follows: potassium nitrate 1900mg/L, ammonium nitrate 1650mg/L,
Potassium dihydrogen phosphate 170mg/L, anhydrous calcium chloride 440mg/L, Magnesium sulfate heptahydrate 370mg/L；Potassium iodide 0.83mg/L, four water
Manganese sulfate 22.3mg/L, boric acid 6.2mg/L, zinc sulphate heptahydrate 8.6mg/L, cobalt chloride hexahydrate 0.025mg/L, copper sulphate pentahydrate
0.025mg/L, Sodium Molybdate Dihydrate 0.25mg/L；Ferrous sulfate 27.85mg/L, sodium ethylene diamine tetracetate 37.25mg/L；Sweet ammonia
Acid 2mg/L, VB1 0.4mg/L, VB6 0.5mg/L, nicotinic acid 0.5mg/L, inositol 100mg/L；Sucrose 30g/L, Phytagel
2.7g/L, Dicamba 2mg/L, Carbenicillin 250mg/L, PPT 3mg/L；
Condition of culture is: cultivate 15-20 days under 25 DEG C of dark conditions.
Wheat anther genetic transforming method the most according to claim 7, goes to be characterised by, the training that described differentiation culture uses
Foster based formulas is: potassium nitrate 1900/L mg, ammonium nitrate 1650mg/L, potassium dihydrogen phosphate 170mg/L, anhydrous calcium chloride 440mg/L,
Magnesium sulfate heptahydrate 370mg/L；Potassium iodide 0.83mg/L, four water manganese sulfate 22.3mg/L, boric acid 6.2mg/L, zinc sulphate heptahydrate
8.6mg/L, cobalt chloride hexahydrate 0.025mg/L, copper sulphate pentahydrate 0.025mg/L, Sodium Molybdate Dihydrate 0.25mg/L；Ferrous sulfate
27.85mg/L, sodium ethylene diamine tetracetate 37.25mg/L；Glycine 2mg/L, VB1 0.4mg/L, VB6 0.5mg/L, nicotinic acid
0.5mg/L, inositol 100mg/L；Vitamin C 100mg/L, 2,4-D 0.5mg/L, sucrose 30g/L, Phytagel 2.7g/L,
Carbenicillin 250mg/L, PPT 3mg/L；
Condition of culture is: under the conditions of 25 DEG C, and illumination every day carries out differentiation culture in 12-14 hour.
9. according to the arbitrary described wheat anther genetic transforming method of claim 7, going to be characterised by, described root culture uses
Prescription of rooting medium be: potassium nitrate 950/L mg, ammonium nitrate 825mg/L, potassium dihydrogen phosphate 85mg/L, anhydrous calcium chloride
220mg/L, Magnesium sulfate heptahydrate 185mg/L；Potassium iodide 0.83mg/L, four water manganese sulfate 22.3mg/L, boric acid 6.2mg/L, seven
Water zinc sulphate 8.6mg/L, cobalt chloride hexahydrate 0.025mg/L, copper sulphate pentahydrate 0.025mg/L, Sodium Molybdate Dihydrate 0.25mg/L；
Ferrous sulfate 27.85mg/L, sodium ethylene diamine tetracetate 37.25mg/L；Glycine 2mg/L, VB1 0.4mg/L, VB6 0.5mg/L,
Nicotinic acid 0.5mg/L, inositol 100mg/L；Naphthalene acetic acid (NAA) 1mg/L, paclobutrazol 3mg/L, sucrose 80g/L, Phytagel
2.7g/L, Carbenicillin 250mg/；
Condition of culture is: when the resistance regeneration bud length that callus differentiates is to about 2-3cm, proceeded to described root media
On carry out strong root and cultivate, when adventitious root grows to 3-4cm, test tube Seedling is transferred in 2-4 DEG C of refrigerator carry out vernalization treatment.
10., according to the arbitrary described wheat anther genetic transforming method of claim 3～6, go to be characterised by, by planting after vernalization
Strain is taken out, and thoroughly removes root culture medium, is transferred in soil cultivate, and when survival and growth to point evil contains the phase, leads under the microscope
Cross Guard Cell Length to differentiate Ploidy of Wheat Pollen Plants, the colchicine of the plant 0.75-1% of guard cell below 65u
Under the conditions of 25 DEG C, soak tillering node with 1.5% dimethyl sulfoxide mixed liquor within 4-5 hour, to obtain doubling homozygous individual.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106717843A (en) * | 2016-11-30 | 2017-05-31 | 襄阳市农业科学院 | A kind of Wheat Haploid embryo gives birth to the cultural method of test tube seedling |
| CN114793904A (en) * | 2022-05-13 | 2022-07-29 | 河北省农林科学院粮油作物研究所 | Induction medium and culture method of wheat anther |
| CN115281082A (en) * | 2022-05-20 | 2022-11-04 | 河北省农林科学院生物技术与食品科学研究所 | Method for improving yield of wheat anther culture green seedling and differentiation culture medium |
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| AMINA REDHA ET AL: "Improvement of green plant regeneration by manipulation of anther culture induction medium of hexaploid wheat", 《PLANT CELL TISS ORGAN CULT》 * |
| 王培等: "提高冬小麦花粉植株诱导率的研究", 《华北农学报》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106717843A (en) * | 2016-11-30 | 2017-05-31 | 襄阳市农业科学院 | A kind of Wheat Haploid embryo gives birth to the cultural method of test tube seedling |
| CN106717843B (en) * | 2016-11-30 | 2019-08-02 | 襄阳市农业科学院 | A kind of cultural method of the raw test tube seedling of Wheat Haploid embryo |
| CN114793904A (en) * | 2022-05-13 | 2022-07-29 | 河北省农林科学院粮油作物研究所 | Induction medium and culture method of wheat anther |
| CN115281082A (en) * | 2022-05-20 | 2022-11-04 | 河北省农林科学院生物技术与食品科学研究所 | Method for improving yield of wheat anther culture green seedling and differentiation culture medium |
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