CN109234311A - The method and classification inoculation apparatus of target gene Agrobacterium are taken in a kind of tobacco leaf inoculation - Google Patents
The method and classification inoculation apparatus of target gene Agrobacterium are taken in a kind of tobacco leaf inoculation Download PDFInfo
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- CN109234311A CN109234311A CN201811349007.9A CN201811349007A CN109234311A CN 109234311 A CN109234311 A CN 109234311A CN 201811349007 A CN201811349007 A CN 201811349007A CN 109234311 A CN109234311 A CN 109234311A
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- 235000002637 Nicotiana tabacum Nutrition 0.000 title claims abstract description 42
- 241000208125 Nicotiana Species 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 30
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 25
- 241000589158 Agrobacterium Species 0.000 title claims abstract description 23
- 239000007788 liquid Substances 0.000 claims abstract description 34
- 241000894006 Bacteria Species 0.000 claims abstract description 20
- 241000196324 Embryophyta Species 0.000 claims abstract description 18
- 239000012530 fluid Substances 0.000 claims abstract description 16
- 239000000725 suspension Substances 0.000 claims abstract description 14
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 13
- 239000001963 growth medium Substances 0.000 claims abstract description 10
- 230000000149 penetrating effect Effects 0.000 claims abstract description 8
- 239000000463 material Substances 0.000 claims abstract description 6
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- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 18
- OJOBTAOGJIWAGB-UHFFFAOYSA-N acetosyringone Chemical compound COC1=CC(C(C)=O)=CC(OC)=C1O OJOBTAOGJIWAGB-UHFFFAOYSA-N 0.000 claims description 18
- 230000001954 sterilising effect Effects 0.000 claims description 12
- 239000002609 medium Substances 0.000 claims description 11
- NJPPVKZQTLUDBO-UHFFFAOYSA-N novaluron Chemical compound C1=C(Cl)C(OC(F)(F)C(OC(F)(F)F)F)=CC=C1NC(=O)NC(=O)C1=C(F)C=CC=C1F NJPPVKZQTLUDBO-UHFFFAOYSA-N 0.000 claims description 11
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L magnesium chloride Substances [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 238000001914 filtration Methods 0.000 claims description 9
- 235000015097 nutrients Nutrition 0.000 claims description 8
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 6
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- 239000005341 toughened glass Substances 0.000 claims description 6
- 244000061176 Nicotiana tabacum Species 0.000 claims description 5
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 4
- 230000003115 biocidal effect Effects 0.000 claims description 4
- 229920001817 Agar Polymers 0.000 claims description 3
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 3
- 229930006000 Sucrose Natural products 0.000 claims description 3
- 239000008272 agar Substances 0.000 claims description 3
- 235000015278 beef Nutrition 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 238000004090 dissolution Methods 0.000 claims description 3
- 239000012154 double-distilled water Substances 0.000 claims description 3
- 239000000284 extract Substances 0.000 claims description 3
- 238000005286 illumination Methods 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- 238000007747 plating Methods 0.000 claims description 3
- 238000003825 pressing Methods 0.000 claims description 3
- 239000005720 sucrose Substances 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- WXCMHFPAUCOJIG-UHFFFAOYSA-N 4'-tert-Butyl-2',6'-dimethyl-3',5'-dinitroacetophenone Chemical compound CC(=O)C1=C(C)C([N+]([O-])=O)=C(C(C)(C)C)C([N+]([O-])=O)=C1C WXCMHFPAUCOJIG-UHFFFAOYSA-N 0.000 claims description 2
- 229910000831 Steel Inorganic materials 0.000 claims description 2
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 claims description 2
- 229940041514 candida albicans extract Drugs 0.000 claims description 2
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- 239000002184 metal Substances 0.000 claims description 2
- 229910052751 metal Inorganic materials 0.000 claims description 2
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- 238000004321 preservation Methods 0.000 claims description 2
- 239000010959 steel Substances 0.000 claims description 2
- 239000000243 solution Substances 0.000 description 15
- 238000005516 engineering process Methods 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 239000013604 expression vector Substances 0.000 description 3
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 description 3
- 229960001225 rifampicin Drugs 0.000 description 3
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical class O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 239000006004 Quartz sand Substances 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- XUWPJKDMEZSVTP-LTYMHZPRSA-N kalafungina Chemical compound O=C1C2=C(O)C=CC=C2C(=O)C2=C1[C@@H](C)O[C@H]1[C@@H]2OC(=O)C1 XUWPJKDMEZSVTP-LTYMHZPRSA-N 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 238000003032 molecular docking Methods 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000005418 vegetable material Substances 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/04—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by injection or suction, e.g. using pipettes, syringes, needles
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Abstract
The present invention is the method and classification inoculation apparatus that target gene Agrobacterium is taken in a kind of inoculation of tobacco leaf.This method passes through: classification inoculation apparatus disinfection；YEB culture medium is prepared；Penetrating fluid is prepared；Cultivate single colonie；Expand culture；Prepare thallus suspension liquid；Tobacco leaf inoculation；8 steps such as culture inoculation tobacco leaf plant are completed, and the present invention is directed to the Agrobacterium that target gene is taken for the inoculation of high-volume tobacco leaf, are quickly and easily high-volume, the tobacco leaf of scale is inoculated with Agrobacterium using novel plant blade inoculation bacterium solution device.Compared with traditional common method, not only increases working efficiency, ensure that effect of inoculation, also save manpower and material.
Description
Technical field
The invention belongs to field of plant cell engineering technology, target gene agriculture is taken in specifically a kind of tobacco leaf inoculation
The method classification inoculation apparatus of bacillus.
Background technique
The Agrobacterium for carrying target gene plant expression vector infects plant host, using mediated by agriculture bacillus, will contain purpose
The plant expression vector of gene is transferred in recipient plant cell, expresses that target gene in recipient cell, this is to convert
A kind of method being widely used in plant cell.To study the target gene in the expression and influence on whole plant, need
The Agrobacterium of the target gene plant expression vector is carried for plant leaf blade inoculation, common inoculation method has smearing to be inoculated with
Method stabs inocalation method and injection inoculation method.Smear inoculation is needed with penetrating fluid suspension thalline, and be added SilwetL-77 and
Quartz sand fine crushing is ground, then thallus suspension liquid is applied on blade with writing brush or other tools.Inocalation method is stabbed, is equally matched
Thallus suspension liquid processed scratches plant leaf blade with syringe needle or other sharp instruments, then thallus suspension liquid is applied to wound inoculation.
Injection inoculation method prepares thallus suspension liquid, draws thallus suspension liquid with the syringe for removing syringe needle, resists vacuum side of blade for bacterium solution
Infiltration is squeezed to entire blade to be inoculated with.
The common method of these types respectively has superiority and inferiority, and smear inoculation is more convenient quickly, but inoculation efficiency is not high；Stab inocalation method
Efficiency is good, but complex steps；Injection inoculation method, efficiency is higher, but takes time and effort consumption material.Using when need according to inoculation
Vegetable material and quantity choose different inoculation methods.
Summary of the invention
The object of the present invention is to provide methods and classification inoculation apparatus that target gene Agrobacterium is taken in a kind of inoculation of tobacco leaf.
Technical scheme is as follows:
A kind of method that target gene Agrobacterium is taken in tobacco leaf inoculation, includes the following steps:
Step 1, classification inoculation apparatus sterilizes；
Step 2, YEB culture medium is prepared, 5g/L beef extract powder, 5g/L peptone, 5g/L sucrose, 1g/L yeast extract,
0.5g/L MgSO4•7H2O, 15g/L agar powder (fluid nutrient medium does not add), add ddH2O dissolution, autoclave sterilization: 121 DEG C
High temperature and pressure 15min is cooled to 60 DEG C or so to culture medium and adds required antibiotic again, later will be in each 100 mm culture dish
30 mL culture mediums are poured into, cooled and solidified is spare；
Step 3, penetrating fluid is prepared, MES 10 mmol/L, MgCl210 mmol/L, 200 μm of ol/L of acetosyringone (AS),
It is ready-to-use；Mother liquor is as follows, MES(1 mol/L), it is prepared with dd water, tune pH value (adding NaOH) to 5.6, filtration sterilization；
MgCl2(1 mol/L), with dd H2O is prepared, filtration sterilization；Acetosyringone (100 mmol/L) weighs 0.3924 g acetyl fourth
Ketone musk (As) is dissolved in dimethyl sulfoxide (DMSO), is settled to 20 ml with DMSO, Bu Shi filter (machine filter film is housed) filtration sterilization,
- 20 DEG C of preservations；
Step 4, single colonie is cultivated, is drawn on the YEB plating medium that the Agrobacterium for carrying target gene is prepared in step 2
Line, in 28 DEG C of dark culturing 16-30 h, until growing single colonie；
Step 5, expand culture, the single colonie that is grown in picking step 4 on YEB culture medium is put in and is prepared in step 1
In YEB fluid nutrient medium, in 28 DEG C, 200 rpm, dark shaken cultivation 16-24 h, until its OD value is 0.6-0.8, it is maximum
OD value is no more than 1.0；
Step 6, thallus suspension liquid is prepared, bacterium solution is placed in 50mL sterile centrifugation tube, and centrifugation (4000 rpm, 15 min, 4
DEG C) supernatant is removed, thallus is collected, thallus is resuspended with the penetrating fluid prepared in step 3, the OD value of bacterium solution is made to reach 1.5-1.8；
Step 7, tobacco leaf is inoculated with, and the thallus suspension liquid prepared in step 6 is fitted into the liquid storage pipe sterilized in step 1
In, tobacco leaf is placed on device pedestal, pressing handle makes the ball syringe needle squeeze the tobacco blade being inoculated on head cause wound,
Ball is squeezed and is lifted in syringe needle, and bacterium solution flows out to leaf dish wound from needle tubing, is lifted handle and is completed once to be inoculated with, according to leaf
Piece size replaces position inoculation；
Step 8, tobacco plant is placed on 25 DEG C by culture inoculation tobacco leaf plant, 65 %-75 % humidity, it is dark for 24 hours after, place into
It is cultivated in following environment: illumination 14 h, 25 DEG C, 65 %-75 % humidity, dark 10 h, 25 DEG C, 65 %-75 % humidity, about 45
Its tobacco plant can bloom.
The classification inoculation apparatus includes pressable handle, inoculation head, pedestal and tempered glass liquid storage pipe with cover, can be pressed
Press two heads of handle respectively with inoculation head and tube chamber welds with base, liquid storage pipe is connect with inoculation head, equipped with several balls on inoculation head
Syringe needle, inoculation head inner hollow and ball syringe needle and liquid storage pipe are connected.
Further, it in step 5, needs to be added Agrobacterium in YEB fluid nutrient medium and carries the anti-of target gene carrier
Property antibiotic.
Further, best in the 4 true leaf period inoculations of tobacco seedling in step 7, more by 4 true leaves all effect of inoculation
It is good.
Compared with prior art, the present invention has the advantages that:
The present invention is directed to the Agrobacterium that target gene is taken for the inoculation of high-volume tobacco leaf, utilizes novel plant blade inoculation bacterium solution
Device is quickly and easily high-volume, the tobacco leaf of scale is inoculated with Agrobacterium.Smear inoculation is more convenient, need to only be incited somebody to action with hairbrush
Prepared suspension bacteria liquid brushes on blade, but inoculation efficiency is not high；It is good to stab inocalation method efficiency, but complex steps,
It needs first to scratch blade, then smears bacterium solution；Injection inoculation method, efficiency is higher, needs certain technology, and infiltrate integrated plate blade
It needs to expend more bacterium solution, injects the repeatedly consumption long period, take time and effort consumption material.Compared with the above common method, utilize
The device and method are the inoculation of high-volume plant leaf blade, can greatly reduce the time consumed by inoculation, reduce the behaviour of experimenter
Make step, reduce the consumption of bacterium solution, and Agrobacterium infects effect after can guarantee inoculation.
Detailed description of the invention
The drawings to be used in the description of the embodiments or prior art will be briefly described below, it is clear that
Ground without creative efforts, can also obtain according to these attached drawings for those of ordinary skill in the art
Obtain other attached drawings.
Fig. 1 is structural schematic diagram of the invention.
In figure, 1- pressable handle, 2- is inoculated with head, 3- pedestal, 4- liquid storage pipe.
Specific embodiment
Below with reference to embodiment, the present invention is described in further detail.
It will be understood to those of skill in the art that the following example is merely to illustrate the present invention, and it should not be regarded as limiting this hair
Bright range.In the examples where no specific technique or condition is specified, described technology or conditions according to the literature in the art
Or it is carried out according to product description.Reagents or instruments used without specified manufacturer is that can be obtained by purchase
Conventional products.
Embodiment 1
The preparation of classification inoculation apparatus: as shown in Figure 1, including pressable handle 1, inoculation head 2, pedestal 3 and tempered glass with cover
Liquid storage pipe 4, pressable handle similar to tweezers structure, two head respectively with inoculation head 2 and pedestal 3 weld, liquid storage pipe with connect
Kind head connection, is inoculated on head and several ball syringe needles is housed, inoculation head inner hollow is connected with ball syringe needle and liquid storage pipe.
Pressable handle 1 is 7.5cm long, and the inoculation head 2 with ball syringe needle is the metal of 3cmx3cm x 0.5cm inner hollow
Square box, the syringe needle that 10x10 root has ball is uniformly distributed with blade contact on one side, and syringe needle long 0.5cm, diameter 0.1cm are inoculated with head
2 are connected to each syringe needle；It connect one side with tempered glass liquid storage pipe 4 with cover and is equipped with interface, it can be with inoculation 2 interface pair of head
It connects.Pedestal 3 is iron and steel material quality, and 3cmx3cmx0.2cm plays the role of support blade.Liquid storage pipe 4 is tempered glass material round tube,
High 5cm, diameter 3cm, facilitate observation bacterium solution usage amount situation, top be equipped with screw thread, can treating plastic tubes lid, bottom be equipped with interface,
The interface of insertion inoculation head 2 realizes docking.
Mode of operation: using before and after the device, can liquid storage pipe, inoculation head and pedestal be dipped in 75% alcohol and is sterilized, taken out
It is reused after drying.It screws a lid on as shown in Figure 1, prepared bacterium solution is contained in liquid storage pipe 4, by interface by liquid storage pipe 4
Insertion inoculation head 2 interconnects, and a hand holds pressable handle 1, and another hand tiles blade on the base 3, presses hand
Handle 1, so that the ball syringe needle on inoculation head 2 squeezes blade, syringe needle punctures blade surface and forms wound, the ball in syringe needle by
Extruding is lifted up, and bacterium solution is immersed in blade wound from syringe needle outflow, and release handle 1 completes a bacterium solution inoculation.According to
Leaf blade size, then moving blade position continue to repeat aforesaid operations inoculation.
Embodiment 2
It is that high-volume tobacco leaf is inoculated with the Agrobacterium for taking tobacco early blossoming gene that the present embodiment, which is based on the device using embodiment 1,
Method, use Agrobacterium for LBA4404 bacterial strain, have streptomysin and rifampicin resistance, carry tobacco early blossoming gene plasmid
With kalamycin resistance, include the following steps:
Step 1, classification inoculation apparatus sterilizes, classification inoculation apparatus is inoculated with as shown in figure 1 first 2 and liquid storage pipe 4 dismantle, be inoculated with head 2 and pedestal 3 and
Liquid storage pipe 4 is immersed in 75% alcohol and sterilizes overnight, dries after taking-up, installs spare.
Step 2, YEB culture medium is prepared, 5g/L beef extract powder, 5g/L peptone, 5g/L sucrose, and 1g/L yeast extracts
Object, 0.5g/L MgSO4•7H2O, 15g/L agar powder (fluid nutrient medium does not add), add ddH2O dissolution.Autoclave sterilization:
121 DEG C of high temperature and pressure 15min are cooled to 60 DEG C or so to culture medium, add 50 mg/L of kanamycins, 50 mg/L of streptomysin
With 25 mg/L of rifampin, about 30 mL culture mediums will be poured into each 100 mm culture dish later, cooled and solidified is spare；
Step 3, penetrating fluid is prepared, 10 mmol/L MES, 10 mmol/L MgCl2, 200 μm of ol/L acetosyringones
(AS), ready-to-use；MES(1 mol/L), it is prepared with dd water, tune pH value (adding NaOH) to 5.6, filtration sterilization；MgCl2(1
Mol/L), with dd H2O is prepared, filtration sterilization；Acetosyringone (100 mmol/L), weighs 0.3924 g acetosyringone
(As) it is dissolved in dimethyl sulfoxide (DMSO), is settled to 20 ml with DMSO, Bu Shi filter (machine filter film is housed) filtration sterilization, -20
DEG C save.
Step 4, single colonie, the YEB plating medium that the Agrobacterium for taking tobacco early blossoming gene is prepared in step 2 are cultivated
Upper scribing line, in 28 DEG C of dark culturing 16-30 h, until growing single colonie；
Step 5, expand culture, the single colonie that is grown in picking step 4 on YEB culture medium is put in and is prepared in step 1
In YEB fluid nutrient medium (50 mg/L of kanamycins, 50 mg/L of streptomysin, 25 mg/L of rifampin), in 28 DEG C, 200
Rpm, dark shaken cultivation 16-24 h, until its OD value is 0.6-0.8, maximum OD value is no more than 1.0；
Step 6, thallus suspension liquid is prepared, bacterium solution is placed in 50mL sterile centrifugation tube, and centrifugation (4000 rpm, 15 min, 4
DEG C) supernatant is removed, collect thallus.Thallus is resuspended with the penetrating fluid prepared in step 3, the OD value of bacterium solution is made to reach 1.5-1.8；
Step 7, tobacco leaf is inoculated with, and the thallus suspension liquid prepared in step 6 is fitted into the liquid storage pipe sterilized in step 1
In, tobacco leaf is placed on device pedestal, pressing handle makes the ball syringe needle squeeze the tobacco blade being inoculated on head cause wound,
Ball is squeezed and is lifted in syringe needle, and bacterium solution flows out to leaf dish wound from needle tubing, is lifted handle and is completed once to be inoculated with, according to leaf
Piece size replaces position inoculation, can all be inoculated with 4 true leaves, best in the 4 true leaf period inoculations of tobacco seedling.
Step 8, tobacco plant is placed on 25 DEG C by culture inoculation tobacco leaf plant, 65 %-75 % humidity, it is dark for 24 hours after, then
It is put into following environment and cultivates: illumination 14 h, 25 DEG C, 65 %-75 % humidity, dark 10 h, 25 DEG C, 65 %-75 % humidity.
In the present embodiment, the tobacco seedlings of tobacco early blossoming gene are inoculated with, 90% or more inoculation is buddingged for plant 45 days or so.
The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent thereof.
Claims (9)
1. a kind of tobacco leaf is inoculated with the method for taking target gene Agrobacterium, characterized by the following steps:
Step 1, classification inoculation apparatus sterilizes；
Step 2, YEB culture medium is prepared, 5g/L beef extract powder, 5g/L peptone, 5g/L sucrose, 1g/L yeast extract,
0.5g/L MgSO4•7H2O, 15g/L agar powder (fluid nutrient medium does not add), add ddH2O dissolution, autoclave sterilization: 121 DEG C
High temperature and pressure 15min is cooled to 60 DEG C or so to culture medium and adds required antibiotic again, later will be in each 100 mm culture dish
30 mL culture mediums are poured into, cooled and solidified is spare；
Step 3, penetrating fluid is prepared, MES 10 mmol/L, MgCl210 mmol/L, 200 μm of ol/L of acetosyringone (AS),
It is ready-to-use；Mother liquor is as follows: MES(1 mol/L), it is prepared with dd water, tune pH value (adding NaOH) to 5.6, filtration sterilization；
MgCl2(1 mol/L), with dd H2O is prepared, filtration sterilization；Acetosyringone (100 mmol/L) weighs 0.3924 g acetyl fourth
Ketone musk (As) is dissolved in dimethyl sulfoxide (DMSO), is settled to 20 ml with DMSO, Bu Shi filter (machine filter film is housed) filtration sterilization,
- 20 DEG C of preservations；
Step 4, single colonie is cultivated, is drawn on the YEB plating medium that the Agrobacterium for carrying target gene is prepared in step 2
Line, in 28 DEG C of dark culturing 16-30 h, until growing single colonie；
Step 5, expand culture, the single colonie that is grown in picking step 4 on YEB culture medium is put in and is prepared in step 1
In YEB fluid nutrient medium, in 28 DEG C, 200 rpm, dark shaken cultivation 16-24 h, until its OD value is 0.6-0.8, it is maximum
OD value is no more than 1.0；
Step 6, thallus suspension liquid is prepared, bacterium solution is placed in 50mL sterile centrifugation tube, and centrifugation (4000 rpm, 15 min, 4
DEG C) supernatant is removed, thallus is collected, thallus is resuspended with the penetrating fluid prepared in step 3, the OD value of bacterium solution is made to reach 1.5-1.8；
Step 7, tobacco leaf is inoculated with, and the thallus suspension liquid prepared in step 6 is fitted into the liquid storage pipe sterilized in step 1
In, tobacco leaf is placed on device pedestal, pressing handle makes the ball syringe needle squeeze the tobacco blade being inoculated on head cause wound,
Ball is squeezed and is lifted in syringe needle, and bacterium solution flows out to leaf dish wound from needle tubing, is lifted handle and is completed once to be inoculated with, according to leaf
Piece size replaces position inoculation；
Step 8, tobacco plant is placed on 25 DEG C by culture inoculation tobacco leaf plant, 65 %-75 % humidity, it is dark for 24 hours after, place into
It is cultivated in following environment: illumination 14 h, 25 DEG C, 65 %-75 % humidity, dark 10 h, 25 DEG C, 65 %-75 % humidity, 45 days
Tobacco plant can bloom.
2. tobacco leaf according to claim 1 is inoculated with the method for taking target gene Agrobacterium, it is characterised in that: step 5
In, it needs to be added Agrobacterium in YEB fluid nutrient medium and carries the resistance antibiotic of target gene carrier.
3. tobacco leaf according to claim 1 is inoculated with the method for taking target gene Agrobacterium, it is characterised in that: step 7
In, it is best in the 4 true leaf period inoculations of tobacco seedling, more preferably by 4 true leaves all effect of inoculation.
4. the classification inoculation apparatus of the method for target gene Agrobacterium, feature are taken in a kind of inoculation of tobacco leaf described in claim 1
It is that the classification inoculation apparatus includes pressable handle, inoculation head, pedestal and tempered glass liquid storage pipe with cover, pressable hand
Respectively with inoculation head and tube chamber welds with base, liquid storage pipe connect two heads of handle with inoculation head, equipped with several ball syringe needles on inoculation head,
It is inoculated with head inner hollow and ball syringe needle and liquid storage pipe is connected.
5. classification inoculation apparatus according to claim 4, it is characterised in that: the inoculation head with ball syringe needle is
The metal square box of 3cmx3cmx 0.5cm inner hollow.
6. classification inoculation apparatus according to claim 4, it is characterised in that: the syringe needle 10x10 root with ball is uniform
It is distributed on inoculation head, syringe needle long 0.5cm, diameter 0.1cm.
7. classification inoculation apparatus according to claim 4, it is characterised in that: the tempered glass liquid storage pipe with cover is circle
Pipe, high 5cm, diameter 3cm, connection one side are equipped with interface, dock with inoculation head interface.
8. classification inoculation apparatus according to claim 4, it is characterised in that: the pedestal is iron and steel material quality, having a size of
3cmx3cmx0.2cm。
9. classification inoculation apparatus according to claim 4, it is characterised in that: the long 7.5cm of pressable handle.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109371057A (en) * | 2018-11-05 | 2019-02-22 | 云南中烟工业有限责任公司 | A kind of method of tobacco high throughput genetic transformation |
| CN109825524A (en) * | 2019-02-26 | 2019-05-31 | 上海迈其生物科技有限公司 | A kind of herbicide basta resistance of mediated by agriculture bacillus imports the method for transformation of tobacco |
| CN111345169A (en) * | 2020-02-25 | 2020-06-30 | 浙江省农业科学院 | Needle-punching type plant germ inoculation method and novel needle-punching type plant germ inoculation equipment |
| CN111937622A (en) * | 2020-08-18 | 2020-11-17 | 广西特色作物研究院 | Inoculation method of citrus canker pathogen |
| CN112048542A (en) * | 2020-07-20 | 2020-12-08 | 中国农业科学院深圳农业基因组研究所 | Inoculation method of puccinia micrantha |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN207193274U (en) * | 2017-06-30 | 2018-04-06 | 四川农业大学 | A kind of microorganism suitable for plant tissue punches inoculator |
| CN207767090U (en) * | 2017-12-13 | 2018-08-28 | 瓜州县瓜州乡农业技术推广协会 | A kind of agricultural edible fungi inoculation device |
-
2018
- 2018-11-13 CN CN201811349007.9A patent/CN109234311A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN207193274U (en) * | 2017-06-30 | 2018-04-06 | 四川农业大学 | A kind of microorganism suitable for plant tissue punches inoculator |
| CN207767090U (en) * | 2017-12-13 | 2018-08-28 | 瓜州县瓜州乡农业技术推广协会 | A kind of agricultural edible fungi inoculation device |
Non-Patent Citations (1)
| Title |
|---|
| 范红军等: "农杆菌介导的大豆反义fad2-1基因转化烟草的研究", 《河南师范大学学报(自然科学版）》, vol. 38, no. 2, pages 152 - 155 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109371057A (en) * | 2018-11-05 | 2019-02-22 | 云南中烟工业有限责任公司 | A kind of method of tobacco high throughput genetic transformation |
| CN109825524A (en) * | 2019-02-26 | 2019-05-31 | 上海迈其生物科技有限公司 | A kind of herbicide basta resistance of mediated by agriculture bacillus imports the method for transformation of tobacco |
| CN111345169A (en) * | 2020-02-25 | 2020-06-30 | 浙江省农业科学院 | Needle-punching type plant germ inoculation method and novel needle-punching type plant germ inoculation equipment |
| CN112048542A (en) * | 2020-07-20 | 2020-12-08 | 中国农业科学院深圳农业基因组研究所 | Inoculation method of puccinia micrantha |
| CN111937622A (en) * | 2020-08-18 | 2020-11-17 | 广西特色作物研究院 | Inoculation method of citrus canker pathogen |
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