CN103074363A - Alfalfa genetic transformation method - Google Patents
Alfalfa genetic transformation method Download PDFInfo
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- CN103074363A CN103074363A CN2012101121822A CN201210112182A CN103074363A CN 103074363 A CN103074363 A CN 103074363A CN 2012101121822 A CN2012101121822 A CN 2012101121822A CN 201210112182 A CN201210112182 A CN 201210112182A CN 103074363 A CN103074363 A CN 103074363A
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Abstract
The present invention relates to an alfalfa genetic transformation method, belongs to the technical field of genetic engineering, and specifically discloses a whole alfalfa seed infection method. The method comprises the following steps: measuring a physiological imbibition curve of mature alfalfa seeds; culturing target gene-containing agrobacterium tumefaciens; carrying out sterilization on the alfalfa seeds; transforming the sterilized alfalfa seeds into the agrobacterium rhizogenes liquid with an OD600 value of 0.8-1.3 to carry out infection for 8-10 h; carrying out co-culture; screening a resistant plant; carrying out rooting and propagation on the resistant plant; and carrying out plant transforming, seedling exercising, atransplantation and the like. According to the present invention, the mature alfalfa seeds are adopted as the experimental material at the first time, the condition that an imbibitions stagnation point without injury is 8-10 h in the alfalfa seed physiological imbibition curve is combined, and the agrobacterium rhizogenes liquid is adopted to soak the seed for 8-10 h at a seed imbibition stage so as to substantially improve transformation efficiency and yield of the transformed plants; and the pressure screening culture medium is adopted to screen the resistant plant, such that an experimental period is shortened by 2-3 months, an experimental process is simplified, identification workload after transformation is reduced, and important significances are provided for theoretical researches and genetic breeding practices of alfalfa genetic engineering.
Description
Technical field
The present invention relates to a kind of method of Genetic Transformation of Alfalfa, belong to the gene engineering field, be specifically related to a kind of alfalfa seed integral body and infect the method for conversion.
Background technology
The grassland is the same with soil, forest, ocean etc., is important strategic resource.The main body on China territory and the main body of TERRESTRIAL ECOSYSTEMS, it is the green ecological barrier of China's area maximum, have and check winds and fix drifting sand, water conservation, conserve water and soil, purify air and the important ecological functions such as biometric safeguard diversity, wash away and the rivers Sediment Siltation reducing earth's surface water and soil, reduce floods hidden danger and have irreplaceable effect.The development grass cultivation is to safeguard the strategic measure of National Ecological Security, the friendly type of built environment society, is the important channel of building modern agriculture, increasing peasants and herdsmen's income; Be conducive to increase the supply of livestock product, reduce the dependence to grain, realize " hiding grain in grass ", enlarge people's food source, improve foodstuff texture, improve the quality of living and level.The development grass cultivation be accelerate the grassland regional development, build a harmonious society in the urgent need to.
Alfalfa is celebrated with " King of Pasture ", and not only grass yield is high, and herbaceous stem is good, and is rich in crude protein, VITAMIN and inorganic salt, and the Amino Acids in Proteins composition is complete, and the animal essential amino acids content is abundant.Gross protein value is 18.8% in the dry-matter, and is higher about 1 times than corn close to half of soya-bean cake, and good palatability can green grass or young crops be raised, ensiling or hay curing.The clover green hay can be throughout the year feeding for domestic animal, and the alfalfa hay of high-quality keeps color and luster dark green, fragrant odour, and quality is soft, and blade does not come off, good palatability.The tender clover of children can feed pig, fowl, rabbit and herbivorous fishes are supplement feeds of protein and VITAMIN.Clover also contains UGF (this is for many years always by the interested problem of scientific and technological circle) except containing the required various nutrients of livestock and poultry.The clover root system is powerful, is good soil conservation plant, and long root nodule on the root, but the nitrogen in the fixed air except the need nitrogen that satisfies self, also can increase the content of Nitrogen In Soils, therefore, also is good green manure plant.These advantages have all determined the critical role of alfalfa in China herbage and even whole grassland industry development.In recent years, ecotope runs down and the cultivation that increases the weight of gradually all to have limited dramatically clover of drought and water shortage, salting of soil or acidifying.Traditional disease and pest problem and a large amount of uses of weedicide are having a strong impact on the yield and quality of clover, have also had a strong impact on ecotope simultaneously, have had a strong impact on development and the ecological construction of national economy.Therefore, improve existing clover cultivar to the tolerance of adverse circumstance and disease and pest and to cultivate the New alfalfa cultivars of degeneration-resistant, Resistant, antiweed extremely urgent.
Breeding parent scarcity of resources, the long restriction by traditional breeding method of cycle are cultivated the good new variety of plant of proterties.Developing rapidly as plant genetics and breeding of plant genetic engineering provides new tool in recent years.The reported first of Genetic Transformation in Higher Plants is in nineteen eighty-three, and since then, transgenic technology is widely used in genetic modification of plants.At present, by this new technology, successfully cultivate many neies variety of plant, wherein much promote and put into production, obtained huge economic and social benefit.Utilize the genetic engineering technique breeding, generally need to by plant tissue culture and specific transgenic method, foreign gene be imported target plant can show the transfer-gen plant of purpose proterties with acquisition, thereby cultivate the good plant new variety.1985, Vash proposed to utilize genetic transfoumation specific foreign gene to be imported the feasibility of herbage on the tissue culture basis for the first time, has established theoretical basis for utilizing the genetic engineering technique Improvement.Since the first in 1986 is succeeded by agriculture bacillus mediated clover conversion, the clover transgenic research is own through having obtained certain progress, comprises the adaptive faculty that strengthens adverse circumstance, to the resistance of various disease and pests, improve nutritional quality, improve digestibility and output etc.In numerous plant genetic transformation method such as Agrobacterium-mediated Transformation method, electric shocking method, microinjection, particle bombardment, one of method that the Agrobacterium-Mediated Transformation method is present most study, frequency of utilization is the highest.The entrained target gene success of Agrobacterium is transferred in the Plant Genome the subsequent experimental important.The explant that conventional agriculture bacillus mediated Genetic Transformation in Higher Plants is often selected is cell, children with meristematic capacity tender tissue, rataria, stem apex and takes preculture etc. to make the competent technology of cell, and these all are conducive to the raising of transformation efficiency.But these method transformation efficiencies are lower, operating process is more loaded down with trivial details, experimental period is also longer.
Summary of the invention
For above-mentioned deficiency, the object of the invention is to overcome the existing difficult point that Genetic Transformation of Alfalfa efficient is low, the transformation period is long and operating process is loaded down with trivial details, the method that provides a kind of alfalfa seed integral body to infect is to improve the transformation efficiency of clover.
The present invention realizes by following technical scheme:
A kind of method of Genetic Transformation of Alfalfa, the method is that the method that adopts alfalfa seed integral body to infect realizes, step comprises: the physiology imbibition curve of 1. surveying ripe alfalfa seed, 2. the agrobacterium tumefaciens that contains target gene is cultivated, 3. alfalfa seed sterilization, 4. in conjunction with the physiology imbibition curve of alfalfa seed, will be transferred to OD through the ripe alfalfa seed of sterilization
600Vibrate in the bacterium liquid of value for the agrobacterium tumefaciens of 0.8-1.3 and infected 8-10 hour, 5. under 25 ℃ of dark conditions, cultivated altogether 3 days, the 6. screening of resistant plant, 7. resistant plant take root and expand numerous, 8. transformed plant hardening and transplanting.
The method of above-described a kind of Genetic Transformation of Alfalfa, wherein:
The step 1. method of the physiology imbibition curve of the ripe alfalfa seed of described survey is: the ripe alfalfa seed of getting 8 parts of equivalent, getting 1 part every 2 hours is dipped in the water, soaked respectively 0,2,4,6,8,10,12,14 hour, the imbibition stagnation point that the record alfalfa seed does not damage is 8-10 hour;
The step 2. described agrobacterium tumefaciens that the contains target gene method of cultivating is: agrobacterium tumefaciens is inoculated on the LB solid medium flat board activation 2 times, and single colony inoculation of this bacterial strain of picking contains 20 ugml to 5 ml afterwards
-1Rifampin and 50 ugml
-1In the LB liquid nutrient medium of kantlex, in temperature 26
oUnder the condition of C, vibration rotating speed 160-200 rpm, cultivated 5-8 hour, then get 1 ml and transfer and contain 20 ugml in 50 ml
-1Rifampin, 50 ugml
-1Kantlex and 110 umolL
-1In the LB liquid nutrient medium of Syringylethanone, in temperature 26
oUnder the condition of C, vibration rotating speed 160-200 rpm, cultivate after 12-16 hour, get 50 ml bacterium liquid and join in the centrifuge tube, place the cryogenic freezing whizzer, in temperature 25
oUnder the condition of C, rotating speed 3500 rpm centrifugal 10 minutes, abandon supernatant liquor, get bacterial sediment, with containing 6 mgL
-16-BA and 110 umolL
-1The resuspended above-mentioned bacterial sediment of the MS liquid nutrient medium of Syringylethanone is to OD
600Value is for subsequent use for 0.8-1.3;
The step 3. method of alfalfa seed sterilization is: select full alfalfa seed, the gauze parcel, after tap water is got express developed totally, with 70% alcohol immersion 2 minutes, soaked 3-5 minute with 0.1% mercuric chloride again, during constantly stir, guarantee that all seed-coat all reach sterilising effect, then with sterilized water washing 5-6 time, prepare to infect usefulness;
Step is described physiology imbibition curve in conjunction with alfalfa seed 4., the method that infects alfalfa seed 8-10 hour is: in conjunction with the physiology imbibition curve of alfalfa seed, to be transferred to through the ripe alfalfa seed of sterilization in the aseptic triangular flask that the agrobacterium tumefaciens bacterium liquid that 2. step prepare is housed, Agrobacterium bacterium liquid measure is as the criterion with firm submergence alfalfa seed, seal bottleneck with sealed membrane, place shaking table, in temperature 26
oUnder the condition of C, vibration rotating speed 160-200 rpm, shaking culture 8-10 hour;
Step 5. described method of cultivating altogether is: after 4. step infects 8-10 hour, takes out from shaking table and place in the Bechtop, outwell bacterium liquid, adding aseptic water washing 3 times places on the common culture medium, under 25 ℃ of dark conditions, cultivates altogether 3 days; Culture medium is to add 6 mgL by the MS liquid nutrient medium altogether
-16-BA and 110 umolL
-1Syringylethanone and 7 gL
-1Agar forms, and pH value 5.80-5.82 forms;
The step 6. method of described screening resistant plant is: will 5. be total to the seed of cultivating through step, and be transferred to and cultivate 1.5-2 month on the pressure screening culture medium, and obtain resistant plant; The pressure screening culture medium is to add 0.2mgL by the 1/2MS solid medium
-16-BA and 1mgL
-1IBA and 250mgL
-1Cef and 75-100 mgL
-1Kan forms;
Step is the taking root and expand numerous method and be of described resistant plant 7.: treat that the resistant plant that step survives after 6. screening is long to 2-3 cm, it is carried out segment expands numerous, be inoculated in and take root on the root media and expand numerous, placing intensity of illumination is 2000 lx, and light application time is to cultivate 30-40 days in the growth cabinet of 12 day/8 night; Root media adds 0.4 gL by the 1/2MS solid medium
-1Gac and 250 mgL
-1Cef and 0-100 mgL
-1Kan forms;
The step 8. method of described transformed plant hardening and transplanting is: when the root of the plant that 7. obtains when step reaches the 10 cm left and right sides, place and uncap 2-3 days in the greenhouse, then with tweezers seedling is pressed from both sides out, with the agar wash clean of the tap water that spends the night with the plant root, be transplanted to Hua Tuzhong, keep soil humidity about 80% after transplanting in 1-2 week, and place the place, cool place of certain illumination, can not be by direct sunlight, after 2 weeks, plant in the flowerpot hot-house culture.
In the method for above-described a kind of Genetic Transformation of Alfalfa, the substratum that adopts in each step is all prepared according to a conventional method with distilled water.Wherein:
Solid LB substratum is by 10gL
-1NaCl, 10gL
-1Peptone, 5gL
-1Yeast extract and 15gL
-1Agar forms, pH value 7.0; Liquid LB substratum does not add agar;
The MS substratum consists of: KNO
31900 mgL
-1, NH
4NO
31650 mgL
-1, CaCl
22H
2O 440 mgL
-1, MgSO
47H
2O 370 mgL
-1, KH
2PO
4170 mgL
-1, H
3BO
36.2 mgL
-1, MnSO
4H
2O 16.9 mgL
-1, ZnSO
47H
2O 8.6 mgL
-1, KCl 0.83 mgL
-1, Na
2MoO
42H
2O 0.25 mgL
-1, CuSO
45H
2O 0.025 mgL
-1, CoCl
26H
2O 0.025 mgL
-1, Na
2EDTA 37.3 mgL
-1, FeSO
47H
2O 27.8 mgL
-1, inositol 20 gL
-1, nicotinic acid 100 mgL
-1, pyridoxine hydrochloride 100 mgL
-1, vitamin 100 mgL
-1, glycine 400 mgL
-1, sucrose 30 gL
-1, pH value 5.80-5.83.
Be total to culture medium: form and see that step 5..
Pressure screening culture medium: form and see that step 6..
Root media: form and see that step 7..
In the method for above-described a kind of Genetic Transformation of Alfalfa:
Step 2. described agrobacterium tumefaciens is the agrobacterium tumefaciens bacterial strain EHA105 that contains target gene gus, and the bacterial strain deposit number of this bacterial strain in national microbial resources storehouse is NKCCMR NK 14.EHA105;
This bacterial strain is used for infecting the preferred OD of bacterial concentration of alfalfa seed
600Value is 1.0.
Step 4. the time period of described alfalfa seed imbibition be 0-10 hour, Agrobacterium is soaked and to infect preferred 9-10 of alfalfa seed time hour.
The present invention has following positive effect:
1, to adopt first ripe alfalfa seed be 8-10 hour as experiment material, the imbibition stagnation point that do not damage in conjunction with the imbibition of alfalfa seed physiology in the present invention, so infected 8-10 hour with agrobacterium tumefaciens bacterium liquid in the Seed imbibition stage, greatly improved transformation efficiency, and the acquisition quantity of transformed plant.
2, through adopting pressure screening culture medium antagonism plant to screen, shorten 2-3 month experimental period, simplified experimentation, reduced the evaluation workload after transforming.
3. method of the present invention practicality simple to operate is compared with microinjection, electric shocking method and other agriculture bacillus mediated method, has clear superiority and breakthrough characteristics, and is all significant to the engineered theoretical investigation of clover and genetic breeding practice.
Description of drawings:
Fig. 1 be the clover that transforms after pressure screening culture medium culturing, all albefactions are defined as transforming not successful alfalfa plants schematic diagram, see Albino Seedling.
Fig. 2 be the clover that transforms after pressure screening culture medium culturing, green seedling survival is arranged, tentatively be defined as transforming successful alfalfa plants schematic diagram, see green seedling.
Fig. 3 is transgenic alfalfa after the pressure screening, and normal growth proves that the foreign gene that changes over to can be to the disadvantageous effect of having grown of transgenic alfalfa.
Fig. 4 is the resistant plant that transgenic alfalfa obtains through the pressure screening, the photo figure in kind of the normal growth situation after the transplanting.
Fig. 5 is the gus gene test figure as a result of not genetically modified alfalfa plants blade, without blue spot, proves that the clover original seed does not contain the gus gene.
Fig. 6 is the gus gene test figure as a result of transgenic alfalfa blade, and blue spot is arranged, and proves that the gus gene successfully is incorporated in the Genomic DNA.
Embodiment
The invention will be further described below in conjunction with case study on implementation:
One, the preparation of substratum and sterilization
1, the preparation of LB liquid nutrient medium: weighing 10 g NaCl, 10 g peptones and 5 g yeast extracts are put in the 1 L triangular flask respectively, add the distilled water constant volume to 1 L, dissolving, and adjust pH to 7.0 with the sealed membrane sealing, adopts 121 afterwards
oC high pressure moist heat sterilization 20 minutes is placed on 4 after the cooling
oIn the C refrigerator, for subsequent use;
2, the preparation of LB solid medium: weighing 10 g NaCl, 10 g peptones, 5 g yeast extracts and 15 g agar are put in the 1 L triangular flask respectively, add the distilled water constant volume to 1 L, dissolving, and adjust pH to 7.0 with the sealed membrane sealing, adopts 121 afterwards
oC high pressure moist heat sterilization 20 minutes is poured in the sterile petri dish, is placed on 4 after the cooling
oIn the C refrigerator, for subsequent use;
3, the preparation of MS substratum:
(1), the preparation of MS substratum mother liquor
1), the preparation of macroelement mother liquor:
1., KNO
3The preparation of mother liquor: take by weighing 190g KNO
3Put in the beaker of 1000 ml, make it to dissolve fully with 800 ml distilled water, the adding distil water constant volume is transferred in the wide-necked bottle room temperature preservation to 1000 ml; It is 100 times than this mother liquor cycles of concentration of working concentration.
2., NH
4NO
3The preparation of mother liquor: take by weighing 330g NH
4NO
3Put in the beaker of 1000 ml, make it to dissolve fully with 800 ml distilled water, the adding distil water constant volume is transferred in the wide-necked bottle room temperature preservation to 1000 ml; It is 200 times than this mother liquor cycles of concentration of working concentration.
3., MgSO47H
2The preparation of O mother liquor: take by weighing 74g MgSO47H
2O puts in the beaker of 1000 ml, makes it to dissolve fully with 800 ml distilled water, and the adding distil water constant volume is transferred in the wide-necked bottle room temperature preservation to 1000 ml; It is 200 times than this mother liquor cycles of concentration of working concentration.
4., KH
2PO
4The preparation of mother liquor: take by weighing 34g KH
2PO
4Put in the beaker of 1000 ml, make it to dissolve fully with 800 ml distilled water, the adding distil water constant volume is transferred in the wide-necked bottle room temperature preservation to 1000 ml; It is 200 times than this mother liquor cycles of concentration of working concentration.
CaCl
22H
2The preparation of O mother liquor: take by weighing 88g CaCl
22H
2O puts in the beaker of 1000 ml, makes it to dissolve fully with 800 ml distilled water, and the adding distil water constant volume is transferred in the wide-necked bottle room temperature preservation to 1000 ml; It is 200 times than this mother liquor cycles of concentration of working concentration.
2), the preparation of micro-mother liquor: take by weighing respectively 6.2 g H
3BO
3, 16.9 g MnSO
4H
2O, 8.6 g ZnSO
47H
2O, 0.83 g KCl, 0.25 g Na
2MoO
42H
2O, 0.025 g CuSO
45H
2O, 0.025 g CoCl
26H
2O puts in the beaker of 1000 ml, makes it to dissolve fully with 800 ml distilled water, and the adding distil water constant volume is transferred in the wide-necked bottle room temperature preservation to 1000 ml; It is 1000 times than this mother liquor cycles of concentration of working concentration.
3), the preparation of mother liquid of iron salt: take by weighing respectively 18.65 g Na
2-EDTA and 13.9 g FeSO
47H
2O puts into respectively 2 500 ml beakers, adds respectively 200 ml distilled water, and heating is also constantly stirred, and makes it to dissolve fully; Cooling mixes 2 kinds of solution, transfers pH to 5.5, and the adding distil water constant volume is transferred in the brown bottle, 4 to 500 ml
oPreserve in the C refrigerator; It is 500 times than this mother liquid concentration cycles of concentration of working concentration.
4), the preparation of the organic mother liquor of B5: take by weighing respectively 10 g inositols, 0.05 g nicotinic acid, 0.05 g pyridoxine hydrochloride, 0.05 g vitamin and 0.2 g glycine, put in the beaker of 500 ml, make it to dissolve fully with 300 mL distilled water, the adding distil water constant volume is to 500 ml, autoclaving, be transferred in the aseptic brown bottle, 4
oPreserve in the C refrigerator; It is 200 times than this mother liquid concentration cycles of concentration of working concentration.
(2), the preparation of MS substratum: draw respectively above-mentioned MS substratum mother liquor: 10 ml KNO3,5 ml NH
4NO
3, 5 ml MgSO47H
2O, 5 ml KH
2PO
4, 5 ml CaCl
22H
2O, 1 ml trace element, 2 ml molysite, 5 ml B5 are organic, join in the 1000 ml triangular flasks that fill 800 ml distilled water, add 30 g sucrose, add the distilled water constant volume to 1 L, fully dissolving, adjust pH is to 5.80-5.83, with the sealed membrane sealing, then adopt 121
oC high pressure moist heat sterilization 20 minutes;
4, the altogether preparation of culture medium: draw respectively above-mentioned MS substratum mother liquor: 10 ml KNO3,5 ml NH
4NO
3, 5 ml MgSO47H
2O, 5 ml KH
2PO
4, 5 ml CaCl
22H
2O, 1 ml trace element, 2 ml molysite, 5 ml B5 are organic, join in the 1000 ml triangular flasks that fill 800 ml distilled water, add 30 g sucrose, 7 g agar, add the distilled water constant volume to 1 L, fully dissolving, adjust pH is to 5.80-5.82, with the sealed membrane sealing, then adopt 121
oC high pressure moist heat sterilization 20 minutes.Take out from pressure kettle, cool off that adding 6 ml concentration are 1mgmL after 10 minutes
-1Aseptic 6-BA, 11 ml concentration are 10 mmolL
-1Aseptic Syringylethanone, fully mixing is poured in the sterile petri dish, is placed on 4 after the cooling
oIn the C refrigerator, for subsequent use;
Two, the genetic transformation of alfalfa seed
1, surveys the physiology imbibition curve of ripe alfalfa seed
Vegetable material is alfalfa seed, get the ripe alfalfa seed of 8 parts (200 every part), got 1 part every 2 hours and be dipped in the water, soaked respectively 0,2,4,6,8,10,12,14 hour, the imbibition stagnation point that the record alfalfa seed does not damage is 8-10 hour;
2, the agrobacterium tumefaciens that contains target gene is cultivated
Select the agrobacterium tumefaciens EHA105 bacterial strain that contains target gene gus, be inoculated on the LB solid medium flat board activation 2 times; Single colony inoculation of this bacterial strain of picking contains 20 ugml to 5 ml afterwards
-1Rifampin and 50 ugml
-1In the LB liquid nutrient medium of kantlex, in temperature 26
oUnder the condition of C, vibration rotating speed 160-200 rpm, cultivated 5 hours; Then getting 1 ml transfers and contains 20 ugml in 50 ml
-1Rifampin, 50 ugml
-1Kantlex and 110 umolL
-1In the LB liquid nutrient medium of Syringylethanone, in temperature 26
oUnder the condition of C, vibration rotating speed 160-200 rpm, cultivated 12 hours; Get 50 ml bacterium liquid and join in the centrifuge tube, place the cryogenic freezing whizzer, in temperature 26
oUnder C, the rotating speed 3500 rpm conditions centrifugal 10 minutes, abandon supernatant liquor, get bacterial sediment; With containing 6mgL
-16-BA and 110 umolL
-1The resuspended above-mentioned bacterial sediment of the MS liquid nutrient medium of Syringylethanone is to OD
600Value is 1.0 for subsequent use;
3, alfalfa seed sterilization
Select full alfalfa seed, after gauze parcel, tap water are got express developed totally, with 70% alcohol immersion 2 minutes, soaked 3-5 minute with 0.1% mercuric chloride again, during constantly stir, guarantee that all seed-coat all reach sterilising effect, then with sterilized water washing 5-6 time, prepare to infect usefulness;
4, infect
In conjunction with the physiology imbibition curve of alfalfa seed, will change over to through the ripe alfalfa seed of sterilization in the agrobacterium tumefaciens bacterium liquid for preparing, Agrobacterium bacterium liquid measure is as the criterion with firm submergence alfalfa seed, seals bottleneck with sealed membrane, places shaking table, in temperature 26
oShaking culture is 10 hours under the condition of C, rotating speed 166 rpm;
5, cultivate altogether
The alfalfa seed after 8-10 hour is infected in immersion, takes out to place in the Bechtop from shaking table, outwells bacterium liquid, adds aseptic water washing 3 times, places on the common culture medium, under 25 ℃ of dark conditions, cultivates altogether 3 days;
6, the screening of resistant plant
After cultivating altogether 3 days, the seed of getting sprouting is transferred to the pressure screening culture medium, and (1/2MS adds 0.2mgL
-16-BA adds 1mgL
-1IBA adds 250mgL
-1Cef adds 75mgL
-1Kan) the upper cultivation 1.5-2 month obtains resistant plant, and such as description of drawings Fig. 1, shown in Figure 2, non-resistant plant among Fig. 1 has resistant plant among Fig. 2;
7, resistant plant takes root and expands numerous
When the resistant plant length that survives after the screening arrives 2-3 cm, it is carried out the segment expansion numerous, (1/2MS adds 0.4 gL to be inoculated in root media
-1Gac adds 250 mgL
-1Cef adds 0-100 mgL
-1Kan) take root on and expand numerously, placing intensity of illumination is 2000 lx, and light application time is to cultivate 30-40 days in the growth cabinet of 12 day/8 night, sees description of drawings Fig. 3.
8, transformed plant hardening and transplanting
When the root of resistant plant reaches the 10 cm left and right sides, place and uncap 2-3 days in the greenhouse, then with tweezers seedling is pressed from both sides out, with the agar wash clean of the tap water that spends the night with the plant root, be transplanted to Hua Tuzhong, keep soil humidity about 80% after transplanting in 1-2 week, and place the place, cool place of certain illumination, can not be by direct sunlight, after 2 weeks, plant in the flowerpot, in the greenhouse, cultivate, see description of drawings Fig. 4.
9, the detection of transfer-gen plant
Whether detect the gus gene by the Gus determination of activity transforms and enters vegetable cell.
Join the Gus staining fluid.
The blade of getting negative control group and conversion group is immersed in the staining fluid, vacuumizes 5 minutes, 37
oC insulation 18 hours.
Take out material, FAA stationary liquid, 95% ethanol respectively decolour 2 times, contrast to feminine gender to be white in color, and see description of drawings Fig. 5.
Naked eyes or microscopically are observed, and the blue point on the white background is the Gus expression sites, sees description of drawings Fig. 6.
The Gus staining fluid consists of: 0.5 mM K
3[Fe (CN)
6], 0.5 mM K
4[Fe (CN)
6], 10 mM Na
2EDTA, 0.1% Triton X-100,0.5 mgmL
-1Paraxin, 0.5 mgmL
-1X-Gluc, 50 mM sodium phosphate buffer (pH7.0) constant volumes, 0.22 um filter membrane suction filtration degerming.
FAA fixedly destainer consists of: 5% formaldehyde, 5% acetic acid, 5% ethanol, distilled water constant volume.
Claims (5)
1. the method for a Genetic Transformation of Alfalfa, the method is that the method that adopts alfalfa seed integral body to infect realizes, step comprises: the physiology imbibition curve of 1. surveying ripe alfalfa seed, 2. the agrobacterium tumefaciens that contains target gene is cultivated, 3. alfalfa seed sterilization, 4. in conjunction with the physiology imbibition curve of alfalfa seed, will be transferred to OD through the ripe alfalfa seed of sterilization
600Value infected 8-10 hour for vibrating in the agrobacterium tumefaciens bacterium liquid of 0.8-1.3,5. under 25 ℃ of dark conditions, cultivated altogether 3 days, the 6. screening of resistant plant, 7. resistant plant take root and expand numerous, 8. transformed plant hardening and transplanting.
2. the method for a kind of Genetic Transformation of Alfalfa according to claim 1 is characterized in that:
The step 1. method of the physiology imbibition curve of the ripe alfalfa seed of described survey is: the ripe alfalfa seed of getting 8 parts of equivalent, getting 1 part every 2 hours is dipped in the water, soaked respectively 0,2,4,6,8,10,12,14 hour, the imbibition stagnation point that the record alfalfa seed does not damage is 8-10 hour;
The step 2. described agrobacterium tumefaciens that the contains target gene method of cultivating is: agrobacterium tumefaciens is inoculated on the LB solid medium flat board activation 2 times, and single colony inoculation of this bacterial strain of picking contains 20 ugml to 5 ml afterwards
-1Rifampin and 50 ugml
-1In the LB liquid nutrient medium of kantlex, in temperature 26
oUnder the condition of C, vibration rotating speed 160-200 rpm, cultivated 5-8 hour, then get 1 ml and transfer and contain 20 ugml in 50 ml
-1Rifampin, 50 ugml
-1Kantlex and 110 umolL
-1In the LB liquid nutrient medium of Syringylethanone, in temperature 26
oUnder the condition of C, vibration rotating speed 160-200 rpm, cultivate after 12-16 hour, get 50 ml bacterium liquid and join in the centrifuge tube, place the cryogenic freezing whizzer, under the 3500 rpm conditions centrifugal 10 minutes, abandon supernatant liquor, get bacterial sediment, with containing 6 mgL
-16-BA and 110 umolL
-1The resuspended above-mentioned bacterial sediment of the MS liquid nutrient medium of Syringylethanone is to OD
600Value is for subsequent use for 0.8-1.3;
Step is described physiology imbibition curve in conjunction with alfalfa seed 4., the method that infects alfalfa seed 8-10 hour is: in conjunction with the physiology imbibition curve of alfalfa seed, to be transferred to through the ripe alfalfa seed of sterilization in the aseptic triangular flask that the agrobacterium tumefaciens bacterium liquid that 2. step prepare is housed, Agrobacterium bacterium liquid measure is as the criterion with firm submergence alfalfa seed, seal bottleneck with sealed membrane, place shaking table, in temperature 26
oC, under the condition of 166 rpm, shaking culture 8-10 hour;
Step 5. described method of cultivating altogether is: after 4. step infects 8-10 hour, outwell bacterium liquid, add aseptic water washing 3 times, place on the common culture medium, under 25 ℃ of dark conditions, cultivated altogether 3 days; Culture medium is to add 6 mgL by the MS liquid nutrient medium altogether
-16-BA and 110 umolL
-1Syringylethanone and 7 gL
-1Agar forms, and pH value 5.80-5.82 forms;
The step 6. method of described screening resistant plant is: will 5. be total to the seed of cultivating through step, and be transferred to and cultivate 1.5-2 month on the pressure screening culture medium, and obtain resistant plant; The pressure screening culture medium is to add 0.2mgL by the 1/2MS solid medium
-16-BA and 1mgL
-1IBA and 250mgL
-1Cef and 75-100 mgL
-1Kan forms;
Step is the taking root and expand numerous method and be of described resistant plant 7.: treat that the resistant plant that step survives after 6. screening is long to 2-3 cm, it is carried out segment expands numerous, be inoculated in and take root on the root media and expand numerous, placing intensity of illumination is 2000 lx, and light application time is to cultivate 30-40 days in the growth cabinet of 12 day/8 night; Root media adds 0.4 gL by the 1/2MS solid medium
-1Gac and 250 mgL
-1Cef and 0-100 mgL
-1Kan forms;
The step 8. method of described transformed plant hardening and transplanting is: when the root of the plant that 7. obtains when step reaches the 10 cm left and right sides, place and uncap 2-3 days in the greenhouse, then with tweezers seedling is pressed from both sides out, with the agar wash clean of the tap water that spends the night with the plant root, be transplanted to Hua Tuzhong, keep soil humidity about 80% after transplanting in 1-2 week, and place the place, cool place of certain illumination, can not be by direct sunlight, after 2 weeks, plant in the flowerpot hot-house culture.
3. the method for a kind of Genetic Transformation of Alfalfa according to claim 2, it is characterized in that: step 2. described Agrobacterium is the agrobacterium tumefaciens bacterial strain that contains target gene gus.
4. the method for a kind of Genetic Transformation of Alfalfa according to claim 2 is characterized in that: 2. step prepares is used for the Agrobacterium bacterial concentration OD that infects
600Value is 0.8-1.3.
5. the method for a kind of Genetic Transformation of Alfalfa according to claim 2 is characterized in that: step 4. the time period of described alfalfa seed imbibition be 0-10 hour, Agrobacterium is soaked and infects the alfalfa seed time is 8-10 hour.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106148396A (en) * | 2015-03-27 | 2016-11-23 | 吉林师范大学 | A kind of method of Semen Tritici aestivi entirety genetic transformation |
| CN106811482A (en) * | 2016-09-26 | 2017-06-09 | 华中农业大学 | Pansy seed infusion method is introduced directly into the genetic transforming method of foreign gene |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105132457B (en) * | 2015-10-19 | 2018-08-03 | 宁夏农林科学院 | A kind of method of fast genetic transformation clover |
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| KENNETH A. FELDMANN ET AL: "Agrobacterium-mediated transformation of germinating seeds of Arabidopsis thMiana: A non-tissue culture approach", 《MOL GEN GENET》 * |
| 徐芳 等: "植物遗传转化的新方法: Floral Dip", 《中国蔬菜》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106148396A (en) * | 2015-03-27 | 2016-11-23 | 吉林师范大学 | A kind of method of Semen Tritici aestivi entirety genetic transformation |
| CN106811482A (en) * | 2016-09-26 | 2017-06-09 | 华中农业大学 | Pansy seed infusion method is introduced directly into the genetic transforming method of foreign gene |
| CN106811482B (en) * | 2016-09-26 | 2020-08-18 | 华中农业大学 | Genetic transformation method for directly introducing exogenous gene by pansy seed soaking method |
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