CN108374020A - A kind of agriculture bacillus mediated soybean transformation in planta method of improvement - Google Patents
A kind of agriculture bacillus mediated soybean transformation in planta method of improvement Download PDFInfo
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- CN108374020A CN108374020A CN201810194837.2A CN201810194837A CN108374020A CN 108374020 A CN108374020 A CN 108374020A CN 201810194837 A CN201810194837 A CN 201810194837A CN 108374020 A CN108374020 A CN 108374020A
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- soybean
- acetosyringone
- agrobacterium
- sillwet
- sucrose
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- 244000068988 Glycine max Species 0.000 title claims abstract description 34
- 235000010469 Glycine max Nutrition 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title claims abstract description 28
- 230000009466 transformation Effects 0.000 title claims abstract description 27
- 230000001404 mediated effect Effects 0.000 title claims abstract description 17
- 241000193830 Bacillus <bacterium> Species 0.000 title claims abstract description 8
- 230000006872 improvement Effects 0.000 title claims abstract description 8
- OJOBTAOGJIWAGB-UHFFFAOYSA-N acetosyringone Chemical compound COC1=CC(C(C)=O)=CC(OC)=C1O OJOBTAOGJIWAGB-UHFFFAOYSA-N 0.000 claims abstract description 30
- 241000589158 Agrobacterium Species 0.000 claims abstract description 19
- 238000002347 injection Methods 0.000 claims abstract description 14
- 239000007924 injection Substances 0.000 claims abstract description 14
- 239000000243 solution Substances 0.000 claims abstract description 12
- 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 abstract description 11
- 229930006000 Sucrose Natural products 0.000 claims abstract description 11
- 239000005720 sucrose Substances 0.000 claims abstract description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 8
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 7
- 210000001672 ovary Anatomy 0.000 claims abstract description 6
- 239000002904 solvent Substances 0.000 claims abstract description 6
- 239000002736 nonionic surfactant Substances 0.000 claims abstract description 5
- 239000007788 liquid Substances 0.000 claims description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 4
- 230000003020 moisturizing effect Effects 0.000 claims description 3
- 238000012545 processing Methods 0.000 claims description 3
- 241000282693 Cercopithecidae Species 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims description 2
- 238000004033 diameter control Methods 0.000 claims description 2
- 238000010438 heat treatment Methods 0.000 claims description 2
- 235000016639 Syzygium aromaticum Nutrition 0.000 claims 2
- 244000223014 Syzygium aromaticum Species 0.000 claims 2
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 claims 2
- 150000002576 ketones Chemical class 0.000 claims 2
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 239000013612 plasmid Substances 0.000 abstract description 6
- 230000002068 genetic effect Effects 0.000 abstract description 4
- 230000009261 transgenic effect Effects 0.000 abstract description 4
- 108091029865 Exogenous DNA Proteins 0.000 abstract 1
- 101710113864 Heat shock protein 90 Proteins 0.000 abstract 1
- 241000196324 Embryophyta Species 0.000 description 15
- 238000006243 chemical reaction Methods 0.000 description 8
- 230000008569 process Effects 0.000 description 5
- 230000008901 benefit Effects 0.000 description 4
- 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 3
- 229930027917 kanamycin Natural products 0.000 description 3
- 239000007921 spray Substances 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 229930189077 Rifamycin Natural products 0.000 description 2
- 108700019146 Transgenes Proteins 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 235000013601 eggs Nutrition 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 230000008929 regeneration Effects 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 229960003292 rifamycin Drugs 0.000 description 2
- HJYYPODYNSCCOU-ODRIEIDWSA-N rifamycin SV Chemical compound OC1=C(C(O)=C2C)C3=C(O)C=C1NC(=O)\C(C)=C/C=C/[C@H](C)[C@H](O)[C@@H](C)[C@@H](O)[C@@H](C)[C@H](OC(C)=O)[C@H](C)[C@@H](OC)\C=C\O[C@@]1(C)OC2=C3C1=O HJYYPODYNSCCOU-ODRIEIDWSA-N 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- ZBMRKNMTMPPMMK-UHFFFAOYSA-N 2-amino-4-[hydroxy(methyl)phosphoryl]butanoic acid;azane Chemical compound [NH4+].CP(O)(=O)CCC(N)C([O-])=O ZBMRKNMTMPPMMK-UHFFFAOYSA-N 0.000 description 1
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 1
- 240000000111 Saccharum officinarum Species 0.000 description 1
- 235000007201 Saccharum officinarum Nutrition 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229960001760 dimethyl sulfoxide Drugs 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 238000012226 gene silencing method Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000002363 herbicidal effect Effects 0.000 description 1
- 239000004009 herbicide Substances 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- 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
Abstract
The invention discloses a kind of agriculture bacillus mediated soybean transformation in planta methods of improvement, it is that will be imported in Agrobacterium tumefaciems containing the plasmid of exogenous DNA, obtain recombinational agrobacterium, after obtained recombinational agrobacterium is suspended with following solution, pass through homemade tool, genetic transformation is carried out into hand-manipulating of needle bundle and injection treatment to the ovary wall of soybean in that afternoon 3~4 point of soybean blossoming:Solvent is water, and solute is nonionic surfactant Sillwet L 77, sucrose and acetosyringone；Wherein, the concentration of volume percent of Sillwet L 77 is 0.1% 0.3%, and the mass percent concentration of sucrose is 5%, and the mass percent concentration of acetosyringone is 0.003%.One Soybean heat shock protein 90 is synthesized in relevant Gene Into Soybean plant using this method, obtains Transgenic soybean plants.
Description
Technical field
The invention belongs to plant genetic engineering fields, and in particular to a kind of agriculture bacillus mediated soybean transformation in planta side of improvement
Method.
Background technology
1988 by the acquisition of Hinchee report Soybean transgenic plants, soybean transgene technology is changed with its orientation
The advantages that short with the breeding time limit is made, starts to develop into a kind of important soybean breeder means.Commonly turn in soybean transgene
Genetic method is agrobacterium-mediated transformation, particle bombardment and pollen tube passage method, and other method for transformation should be used less.
Agrobacterium-mediated transformation is to use a kind of wide transformation of soybean method, its advantage is that easy to operate, research expenditure
Relatively low, transfer-gen plant usually exists in the form of singly copy or low-copy, and descendant inheritting performance is relatively stablized.But the disadvantage is that turning
Change frequency it is relatively low, either plant regeneration or conversion process have stronger genotype-independent, be restricted factor compared with
It is more.
Particle bombardment is also to use a kind of wide genetic transforming method, its advantage is that the influence of acceptor material is smaller,
It is not limited by recipient genotypes and bombardment target tissue, transformation efficiency is relatively high, easy to operate, and degree of controllability is high.But also have one
Fixed disadvantage, for example, research expenditure costly, conversion ratio it is relatively low, reset frequent occurrence in conversion process and high copy be inserted into it is existing
As inactivation and the descendant inheritting rule for being easy to cause foreign gene are disorderly, make later stage screening that can express target gene transgenosis
Plant is relatively difficult, and copy number is high in the plant genome, easily causes gene silencing.
Pollen tube passage method has broken away from the limitation of receptor regeneration condition, can be directly obtained after the completion of conversion process transgenosis by
The seed of body reduces the time cycle, but because its transformation efficiency is extremely low, operation requires height, and have conversion target confusion etc.
Problem, so using less.
Therefore, searching is a kind of carrying out soybean transformation work in scale under field condition, and operation requires low conversion to imitate
The high method for transformation of rate is particularly important.
Invention content
The object of the present invention is to provide a kind of agriculture bacillus mediated soybean transformation in planta methods of improvement.
A kind of agriculture bacillus mediated soybean transformation in planta method of improvement, it is characterised in that specifically comprise the following steps：
The Agrobacterium tumefaciems for choosing carrying target gene is cultivated to OD600Value removes supernatant after 0.8-1.2, centrifugation, is added
Isometric solution A, mixing obtain Agrobacterium-mediated Transformation liquid with the supernatant that discards；Choose that afternoon 3~4 point of soybean blossoming
Injection Agrobacterium-mediated Transformation liquid processing is carried out to the ovary wall of soybean, jacketed polybag moisturizing after the completion of operation maintains to next day to insult
Morning 5:00~6:00；Wherein, for the solution A using water as solvent, solute is nonionic surfactant Sillwet L-77, sugarcane
Sugar and acetosyringone；Wherein, the concentration of volume percent of Sillwet L-77 is 0.1%-0.3%, the quality percentage of sucrose
Specific concentration is 3%-5%, and the mass percent concentration of acetosyringone is 0.001-0.004%, and acetosyringone is with appropriate two
Methyl sulfoxide dissolves.
The method of the present invention be suitable for all soybean varieties described in the preferred Williams 82 of soybean varieties, Nan Nong 88-31,
Monkey hair.
The injection Agrobacterium-mediated Transformation liquid is injected using syringe or is handled using following self-control injection tool；Self-control note
The tool of penetrating be by commercially available rubber head suction pipe, by the heating means such as burning at water suction, by nozzle diameter control within 0.5 millimeter,
Its structure is substantially that one end is commercially available rubber head dropper, and one end is the injection port that diameter is less than 0.5 millimeter, and intermediate length is unlimited.
The solution A preferably using water as solvent, solute be nonionic surfactant Sillwet L-77, sucrose and
Acetosyringone；Wherein, the concentration of volume percent of Sillwet L-77 is 0.1%-0.3%, and the mass percent of sucrose is dense
Degree is 5%, and the mass percent concentration of acetosyringone is 0.003%, the appropriate dmso solution of acetosyringone.
Advantageous effect
Compared with prior art, the present invention having the following advantages that and good effect：
It, can scale progress transformation experiment 1. entire conversion process can be completed under field condition.
2. conversion process can shorten experimental period 3-5 months departing from the limitation of tissue cultures.
3. being directly to infect target with fertilized eggs, transgenosis T1 can be directly obtained for seed or chimera transgenic seed.
4. Agrobacterium injection is directly entered ovary wall by self-made tool, Agrobacterium can be effectively increased and infect fertilized eggs
Efficiency.
5. experimental implementation is easy, difficulty is low.
Description of the drawings
Fig. 1 is that soybean floral organ ovary wall needle is pricked and injection position schematic diagram
Fig. 2 is self-control Agrobacterium injection tool
Fig. 3 is plasmid pCambia3301:GmHSP90A2 collection of illustrative plates
Fig. 4 is that genetically engineered soybean T1 sprays the selection result for bar
Fig. 5 genetically engineered soybeans T2 is for Bar ELISA test strip results
Fig. 6 is that genetically engineered soybean T1 detects (M for Bar resistant plants PCR：Standard molecular weight；1：Negative control；2：It is positive right
According to；3~8:Bar resistant plants)
Specific implementation mode
Method therefor is conventional method unless otherwise instructed in following embodiments.
Following embodiments are intended to further illustrate the present invention, are not intended to limit the present invention.
Embodiment 1
(1) soybean planting
Soybean varieties Williams 82 is planted with mid-June in the big Tanaka in Transgenic studies area, with every two row every a line
Method plantation, enter full-bloom stage mid-August current year, begin to function as experiment receptor.
(2) Agrobacterium is cultivated
pCambia3301:GmHSP90A2 plasmid maps are shown in Fig. 3, are the plasmid that sets out, plasmid T- with pCambia3301
Target gene is carried on DNA, Bar screening-genes, wherein target gene GmHSP90A2 (SEQ ID NO.1) are located at restriction enzyme site
It is between BamH1 and Xba1.Agrobacterium tumefaciens strain is EHA105.By be stored in -80 DEG C contain pCambia3301 plasmids
EHA105 bacterial strains activated, scribing line culture the gentle 50mg/L kanamycins of rifamycin antibiotic containing 50mg/L YEB solids
On tablet, 2d is cultivated at 28 DEG C, then picking single bacterium, which is fallen in sterile 1.5ml centrifuge tubes, (is added 1ml to contain in centrifuge tube
The YEB fluid nutrient mediums of the gentle 50mg/L kanamycins of 50mg/L rifamycin antibiotics) shake culture 1d, 28 DEG C, 200rpm.And
PCR detections are carried out afterwards, choose the bacterium solution of the PCR positives as mother liquor.
YEB fluid nutrient mediums are prepared, 50mg/L Antibiotic Rifampicins, 50mg/L kanamycins and 1ml/L mother liquors, shake is added
It swings and cultivates 1d, 28 DEG C, 200rpm, until OD600Value be 1.0.4000rpm centrifuges 10min, removes supernatant, the agriculture of equivalent is added
(solvent is water to Agrobacterium-transformation liquid, and solute is nonionic surfactant Sillwet L-77, sucrose and acetosyringone；Its
In, the concentration of volume percent of Sillwet L-77 is 0.2%, and the mass percent concentration of sucrose is 5%, acetosyringone
Mass percent concentration be 0.003%, the appropriate dmso solution of acetosyringone.)
(3) time at or so selection 3~4 o'clock of afternoon, the Agrobacterium-mediated Transformation liquid that will have been configured pass through self-made tool (figure
2) it is pierced into soybean ovary wall (Fig. 1), injection Agrobacterium is infected.
(4) jacketed polybag moisturizing after the completion of dip dyeing operation, maintains to remove bag to morning next day.After seed maturity, harvest
It dries to get T1 for seed.
(4) T1 of acquisition is seeded in for 10000, seed or more in greenhouse, in Seedling Stage by the screening of transfer-gen plant
Herbicide spraying Basta, primary, continuous spray 6 times was sprayed every 5 days, and counting survival behind complete finally to spray 30 days plants
Strain is 39 plants total, collects blade extraction DNA, carries out transgenosis PCR detections, find positive plant 17 plants (Fig. 5).The present invention's
Transformation of soybean efficiency is 0.17% or so.Progeny plants Bar ELISA test strip results confirm genetic transformation event success (Fig. 4),
Obtain heritable transfer-gen plant.
Sequence table
<110>Agricultural University Of Nanjing
<120>A kind of agriculture bacillus mediated soybean transformation in planta method of improvement
<150> 2017114590192
<151> 2017-12-28
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2097
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
atggcggacg ctgaaacctt cgctttccag gctgagatca accagcttct cagcctcatc 60
atcaacacct tctacagcaa caaggagatt ttcttgcgtg aactcatcag caacgcctct 120
gatgctttgg ataagattcg atttgagagt ttaaccgaca agagcaagct cgatgctcag 180
ccagagctct tcattcgtct tgtgcccgac aaggccaaca agactctttc cgttattgac 240
agcggcattg gaatgaccaa agcagatttg gtgaacaact tggttaccat tgcgaggtcc 300
gggaccaagg agttcatgga ggcattgcag gctggtgctg atgtaagcat gattgggcag 360
ttcggtgtgg ggttctactc tgcttacctc gttgcagaga aggtcattgt taccaccaag 420
cacaatgatg atgaacagta catttgggag tctcaggccg gtggctcttt cactgttact 480
agggacaccg atggcgagca actcggaagg ggaaccaaga tgaccctctt cctcaaggaa 540
gatcagttgg agtacttgga ggagagaagg atcaaagatc tggtgaagaa gcactctgaa 600
ttcattagct atcctattta cctatggact gagaagacca ccgagaaaga aatcagcgat 660
gatgaagatg atgagccaaa gaaggaggaa gaaggggacg ttgaggatgt tgacgaggac 720
aaggagaagg attccaagaa aaagaagaag atcaaggagg tgtctcatga gtggcaactc 780
atcaacaagc agaaaccaat ttggctgcga aagccagaag agatcaccaa ggatgaatac 840
gcttctttct acaagagcct caccaatgac tgggaagaac acttggctgt gaaacatttc 900
tctgttgagg gccagcttga gttcaaggcc attctctttg tgccaaagag ggcacccttt 960
gatttgtttg acaccaggaa gaagatgaac aacatcaaac tttatgtcag gagggtgttc 1020
atcatggaca actgtgagga gctcattccc gagtacctcg gatttgtgaa gggtgttgtc 1080
gactccgatg acttgcccct caacatttct cgtgagctgc tgcagcagaa caagatcctg 1140
aaggtgatca ggaagaatct tgtgaagaag tgcattgaga tgttcaatga aattgcggag 1200
aacaaggaag actacaacaa gttctatgat gctttttcga agaatttgaa gttgggtatc 1260
cacgaggata gccagaacag gtccaagttg gctgatttgc tcagatacca ctcaacaaag 1320
agtggagacg aattgacaag cttgaaggac tatgttacac gaatgaagga gggccagaag 1380
gacatttact acattacagg agagagcaag aaggcagtgg agaactctcc tttcttggag 1440
agactcaaga agaagggcta tgaagttctc ttcatggtgg atgcaattga tgagtatgca 1500
gttgggcaac tcaaggaata cgatggcaag aaattggttt cagccacaaa ggaagggttg 1560
aagctggatg atgagactga ggaggagaaa aagaagaaag aggataagaa gaagtcattt 1620
gatgagctct gcaaggtcat caaggaaatt ctgggagaca aagtggagaa ggttgttgtc 1680
tctgacagaa ttgttgactc tccttgctgt ttggtgaccg gtgaatacgg atggagtgca 1740
aacatggaaa ggatcatgaa ggctcaggct ctgagagaca gcagcatgag tggctacatg 1800
tctagcaaga agacaatgga gatcaaccct gacaatggta tcatggagga gttgaggaag 1860
agggctgaag ctgacaagaa tgataagtct gtgaaggatc ttgtgttgct tctctttgag 1920
actgcccttt tgacttcagg tttcagcctt gatgatccca acacatttgc ttcgaggatt 1980
cacaggatgt tgaagttggg tcttagcatt gatgaggatg acaatggtgg agacgatgtt 2040
gatatgcccc cattggaaga ggacggtgct gaggagagca agatggagga agtggac 2097
Claims (4)
1. a kind of agriculture bacillus mediated soybean transformation in planta method of improvement, it is characterised in that specifically comprise the following steps：
The Agrobacterium tumefaciems for choosing carrying target gene is cultivated to OD600Value removes supernatant after 0.8-1.2, centrifugation, be added with it is upper
The isometric solution A of clear liquid, mixing obtain Agrobacterium-mediated Transformation liquid；Choose ovary of that afternoon 3~4 point to soybean of soybean blossoming
Wall carries out injection Agrobacterium-mediated Transformation liquid processing, and jacketed polybag moisturizing after the completion of operation was maintained to morning next day 5:00~6:00；
Wherein, for the solution A using water as solvent, solute is nonionic surfactant Sillwet L-77, sucrose and acetyl cloves
Ketone；Wherein, the concentration of volume percent of Sillwet L-77 is 0.1%-0.3%, and the mass percent concentration of sucrose is 3%-
5%, the mass percent concentration of acetosyringone is 0.001-0.004%, the appropriate dmso solution of acetosyringone.
2. the method according to claim 1, it is characterised in that the soybean varieties are Williams 82, Nan Nong 88-31, monkey
Sub- hair.
3. the method according to claim 1, it is characterised in that the injection Agrobacterium-mediated Transformation liquid is injected or adopted using syringe
With self-control injection tool processing；The self-control injection tool is to pass through and the heating side such as burn commercially available rubber head suction pipe at water suction
Method, by nozzle diameter control within 0.5 millimeter, structure is substantially that one end is commercially available rubber head dropper, and one end is that diameter is small
In 0.5 millimeter of injection port, intermediate length is unlimited.
4. the method according to claim 1, it is characterised in that the solution A using water as solvent, for non-ionic surface live by solute
Property agent Sillwet L-77, sucrose and acetosyringone；Wherein, the concentration of volume percent of Sillwet L-77 is 0.1%-
0.3%, the mass percent concentration of sucrose is 5%, and the mass percent concentration of acetosyringone is 0.003%, acetyl cloves
The appropriate dmso solution of ketone.
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