CN113604498A - Stylosanthes guianensis hairy root induction method - Google Patents
Stylosanthes guianensis hairy root induction method Download PDFInfo
- Publication number
- CN113604498A CN113604498A CN202110879039.5A CN202110879039A CN113604498A CN 113604498 A CN113604498 A CN 113604498A CN 202110879039 A CN202110879039 A CN 202110879039A CN 113604498 A CN113604498 A CN 113604498A
- Authority
- CN
- China
- Prior art keywords
- stylosanthes guianensis
- stylosanthes
- guianensis
- culture
- culturing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241000412329 Stylosanthes hispida Species 0.000 title claims abstract description 88
- 238000000034 method Methods 0.000 title claims abstract description 30
- 230000006698 induction Effects 0.000 title claims abstract description 21
- 241000589156 Agrobacterium rhizogenes Species 0.000 claims abstract description 36
- 238000012258 culturing Methods 0.000 claims description 21
- 239000001963 growth medium Substances 0.000 claims description 18
- 238000005286 illumination Methods 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 239000008223 sterile water Substances 0.000 claims description 16
- 238000003501 co-culture Methods 0.000 claims description 10
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 claims description 10
- 230000035784 germination Effects 0.000 claims description 9
- 230000001954 sterilising effect Effects 0.000 claims description 7
- 229930006000 Sucrose Natural products 0.000 claims description 6
- 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 6
- 230000003115 biocidal effect Effects 0.000 claims description 6
- 230000001939 inductive effect Effects 0.000 claims description 6
- 239000002609 medium Substances 0.000 claims description 6
- 239000005720 sucrose Substances 0.000 claims description 6
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 claims description 5
- 229910004619 Na2MoO4 Inorganic materials 0.000 claims description 5
- 239000003242 anti bacterial agent Substances 0.000 claims description 5
- 229940088710 antibiotic agent Drugs 0.000 claims description 5
- 239000001110 calcium chloride Substances 0.000 claims description 5
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 5
- 229910052927 chalcanthite Inorganic materials 0.000 claims description 5
- 229910052564 epsomite Inorganic materials 0.000 claims description 5
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 claims description 5
- 239000011684 sodium molybdate Substances 0.000 claims description 5
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 4
- 239000004471 Glycine Substances 0.000 claims description 4
- 238000005520 cutting process Methods 0.000 claims description 4
- 239000006870 ms-medium Substances 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- 230000005059 dormancy Effects 0.000 claims description 3
- 230000003213 activating effect Effects 0.000 claims description 2
- FPPNZSSZRUTDAP-UWFZAAFLSA-N carbenicillin Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)C(C(O)=O)C1=CC=CC=C1 FPPNZSSZRUTDAP-UWFZAAFLSA-N 0.000 claims description 2
- 229960003669 carbenicillin Drugs 0.000 claims description 2
- 230000000249 desinfective effect Effects 0.000 claims description 2
- 208000015181 infectious disease Diseases 0.000 abstract description 6
- 238000011160 research Methods 0.000 abstract description 5
- 241000196324 Embryophyta Species 0.000 description 15
- 230000009261 transgenic effect Effects 0.000 description 14
- 230000001580 bacterial effect Effects 0.000 description 10
- 230000009466 transformation Effects 0.000 description 10
- 238000001514 detection method Methods 0.000 description 8
- 239000000463 material Substances 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 239000000203 mixture Substances 0.000 description 7
- 241000589158 Agrobacterium Species 0.000 description 4
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 3
- 239000005708 Sodium hypochlorite Substances 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 239000012869 germination medium Substances 0.000 description 3
- 238000009630 liquid culture Methods 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 3
- 244000153158 Ammi visnaga Species 0.000 description 2
- 235000010585 Ammi visnaga Nutrition 0.000 description 2
- 241000045403 Astragalus propinquus Species 0.000 description 2
- 240000001829 Catharanthus roseus Species 0.000 description 2
- 101800004637 Communis Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 244000068988 Glycine max Species 0.000 description 2
- 235000010469 Glycine max Nutrition 0.000 description 2
- 239000007836 KH2PO4 Substances 0.000 description 2
- 244000046052 Phaseolus vulgaris Species 0.000 description 2
- 241000131460 Plectranthus Species 0.000 description 2
- 229920001938 Vegetable gum Polymers 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 235000006533 astragalus Nutrition 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 210000001808 exosome Anatomy 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 230000002335 preservative effect Effects 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 235000013619 trace mineral Nutrition 0.000 description 2
- 239000011573 trace mineral Substances 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 241001297689 Candidatus Glassbacteria Species 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 241000522190 Desmodium Species 0.000 description 1
- 206010020649 Hyperkeratosis Diseases 0.000 description 1
- 101150062179 II gene Proteins 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 241001495120 Stylosanthes Species 0.000 description 1
- 241000412326 Stylosanthes erecta Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000002390 adhesive tape Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 150000001361 allenes Chemical class 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- -1 bar Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 230000002363 herbicidal effect Effects 0.000 description 1
- 239000004009 herbicide Substances 0.000 description 1
- 238000011177 media preparation Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 239000011859 microparticle Substances 0.000 description 1
- 238000004161 plant tissue culture Methods 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Inorganic materials [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 238000009331 sowing Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Images
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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a stylosanthes guianensis hairy root induction method which comprises the steps of selection and culture of agrobacterium rhizogenes, obtaining of stylosanthes guianensis explants, infection of the stylosanthes guianensis explants, induction of the stylosanthes guianensis hairy roots, culture of the hairy roots in a sterile manner and propagation, wherein the inductivity of the hairy roots reaches 58.3%. The stylosanthes guianensis hairy root induction method provides important technical support for molecular biology research of stylosanthes guianensis, and has a wide application prospect.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a stylosanthes guianensis hairy root induction method.
Background
Stylosanthes guianensis is perennial legume grass mainly distributed in tropical and subtropical areas, has high yield and good quality, and is mainly used for animal feed, forest land greening and the like. Stylosanthes guianensis is an excellent pioneer crop for improving acid soil.
In nature, Agrobacterium rhizogenes (Agrobacterium rhizogenes) can stably integrate rol genes inducing hairy roots into a host plant genome through T-DNA of Ri plasmid to generate hairy roots, and can simultaneously integrate exogenous genes carried in Ti plasmid into the plant genome together, thereby realizing co-transformation. The use of Agrobacterium rhizogenes to obtain transgenic plant hairy roots has been realized in a variety of plants. For example, hairy roots obtained from plants such as kidney bean (Phaseolus vulgaris), soybean (Glycine max), Astragalus membranaceus (Astragalus membranaceus), and vinca rosea (Catharanthus roseus) are widely used for obtaining root secondary metabolites and studying gene functions.
At present, the transgenic material of the stylosanthes guianensis is mainly obtained by a whole plant transformation method. In 1987, Manners et al succeeded in introducing NTP II gene into Stylosanthes guianensis successfully and obtaining whole transgenic Stylosanthes guineensis by Agrobacterium tumefaciens infection for the first time (Transformation of Stylosanthes spp. using Agrobacterium tumefaciens [ J ]. Plant Cell Reports, 1987). In addition, Manners infected the stems and leaves of Stylosanthes guianensis with Agrobacterium rhizogenes to obtain 23% complete transgenic material. Sarria et al transferred the binary vector epGV1040 to Stylosanthes guianensis with Agrobacterium tumefaciens and found that the foreign gene was integrated into the genome of the transgenic Plant in a single copy form (Agrobacterium-mediated transformation of Stylosanthes guianensis and transformation of transgenic plants [ J ]. Plant Science, 1994). In addition to the method using Agrobacterium mediation to obtain transgenic material, Quecini et al used the particle gun method to introduce Be2S1 gene into Stylosanthes guianensis and established a particle gun method genetic Transformation system (Microparticle Bombardent of Stylosantes guianensis: Transformation parameters and expression of a methionine-rich 2Salbumin gene [ J ]. Plant, Tissue and organic Culture, 2006). However, in China, until recently, a genetic transformation system for transforming stylosanthes guianensis is further established and optimized, and the transformation system of stylosanthes guianensis and the like is optimized, so that transgenic stylosanthes guianensis containing genes such as bar, hpt, npt II and the like are successfully obtained, and researches also find that the hypocotyl callus induction rate and the differentiation rate of stylosanthes guianensis seedlings are higher than those of other parts, so that the stylosanthes guianensis is an ideal explant material part (transgenic cold resistance improvement research [ D ] of stylosanthes university of agriculture, university, Washington, 2012). In 2012, the plectranthus communis et al used 5 st stylosanthes guianensis as a material, and utilized agrobacterium infection to successfully introduce allene oxidase gene (AOC) into the stylosanthes guianensis to obtain a whole transformation material (the plectranthus communis, guo jian chun, prune et al. In addition, further application and improvement of the above-mentioned whole plant transformation system of stylosanthes guianensis have been reported.
However, the method for transforming the whole plant to obtain the transgenic material has the defects of relatively complicated transgenic process, relatively low transgenic efficiency, excessively long transformation time and the like, and is not favorable for researching the gene function of the stylosanthes guianensis. Therefore, the invention of the stylosanthes guianensis hairy root induction method has important significance for researching the gene function of the stylosanthes guianensis, but no report is found about the induction of the stylosanthes guianensis hairy root at present.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a stylosanthes guianensis hairy root induction method.
The above object of the present invention is achieved by the following technical solutions:
a stylosanthes guianensis hairy root induction method comprises the following steps:
s1, selection and culture of agrobacterium rhizogenes: activating and culturing an agrobacterium rhizogenes strain MSU440 into an agrobacterium rhizogenes liquid for later use;
s2, obtaining a stylosanthes guianensis explant: cutting the cotyledon node and/or cotyledon of the germinated stylosanthes guianensis seedling to be an explant;
s3, infecting stylosanthes guianensis explants: co-culturing the stylosanthes guianensis explant in the step S2 with the agrobacterium rhizogenes liquid in the step S1;
s4, inducing the styloid roots of stylosanthes guianensis: co-culturing the explant of the stylosanthes guianensis infected in the step S3 in sterile water for 4-6 days, transferring the explant to sterile filter paper soaked in the co-culture solution for co-culturing for 12-15 days, and preliminarily obtaining hairy roots at the cut;
s5, hairy root bacteria-removing culture and propagation: and (3) after the hairy roots grow to be more than 2cm, washing the hairy root transformants with sterile water containing antibiotics for three times, transferring the hairy root transformants into an MS culture medium containing antibiotics, subculturing for a plurality of times until the agrobacterium rhizogenes is completely killed, and finally transferring the hairy roots into 1/2MS culture medium to propagate the hairy roots.
Preferably, the Agrobacterium rhizogenes bacterial solution obtained in the step S1 is obtained by absorbing 200-300 μ L of activated Agrobacterium rhizogenes strain MSU440 seed bacterial solution, inoculating into 5-10 mL YEP liquid culture medium, and performing shake culture at 26-28 ℃ and 170-190 r/min until OD is 0.5-0.6.
Preferably, the germinating in step S2 is: sterilizing and disinfecting the stylosanthes guianensis seeds after dormancy breaking, and placing the stylosanthes guianensis seeds in a germination culture medium for culturing and germinating; the culture temperature is 22-24 ℃, and the illumination intensity is 110-130 mu mol.m-2·S-1The illumination time is 15-17 hours per day, and the relative humidity is 55-65%.
Further preferably, the germination medium is a 1 × MS medium.
Preferably, the stylosanthes guianensis explant is a stylosanthes guianensis seedling cotyledon which germinates for 2 days and has a hypocotyl of 1 mm.
Preferably, the co-culture solution of step S4 is composed of: 0.4125 g.L-1NH4NO3、0.48g·L-1KNO3、0.11g·L-1CaCl2·2H2O、0.0925g·L-1MgSO4·7H2O、0.0425g·L-1KH2PO4、0.2075mg·L-1KI、1.54mg L-1H3BO3、5.56mg·L-1MnSO4·4H2O、2.65mg·L-1ZnSO4.7H2O、0.0625mg·L- 1Na2MoO4·2H2O、0.00625mg·L-1CuSO4·5H2O、0.00625mg·L-1CoCl2·6H2O、9.325mg·L-1Fe-EDTA、25mg·L-1Inositol, 0.5 mg.L-1Glycine; 0.5% sucrose. Namely, 25 mg. L was added to 1/4 XMS medium-1Inositol, 0.5 mg.L-1Glycine and 0.5% sucrose.
Preferably, the step S4 is implemented by culturing in sterile water at 24-26 ℃, with the illumination intensity of 39000-39100 lx, the illumination time of 15-17 hours per day and the relative humidity of 45-55% for co-culturing 4-6; the temperature of the stylosanthes guianensis explant and the co-culture solution are 22-24 ℃, the illumination intensity is 39000-39100 lx, and the illumination co-culture lasts for 12-15 days.
Preferably, the antibiotic in the antibiotic-containing sterile water and the antibiotic-containing MS medium in the step S5 is carbenicillin (Carb) with a concentration of 4-6 g/L.
The stylosanthes guianensis seed disclosed by the invention selects different varieties of germplasm resources according to different requirements, and preferably, the stylosanthes guianensis seed selected by the invention is RY 2.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a stylosanthes guianensis hairy root induction method which comprises the steps of selection and culture of agrobacterium rhizogenes, obtaining of stylosanthes guianensis explants, infection of the stylosanthes guianensis explants, induction of the stylosanthes guianensis hairy roots, culture and propagation of the hairy roots in a germ-free mode, wherein the inductivity of the hairy roots reaches 58.3%, and an important technical support is provided for molecular biology research of the stylosanthes guianensis.
Drawings
FIG. 1 is a schematic diagram of the process of inducing the hairy roots of stylosanthes guianensis according to the invention. A. Obtaining a stylosanthes guianensis explant; B. culturing agrobacterium rhizogenes; C. the agrobacterium rhizogenes infects the stylosanthes guianensis seedling; D. co-culturing the stylosanthes guianensis explant and the agrobacterium rhizogenes in sterile water; E. inducing the hair roots of stylosanthes guianensis; F. the hairy root shape of stylosanthes guianensis. The scale is 2 cm.
FIG. 2 shows the results of transgenic hairy root detection.
FIG. 3 shows the results of the test for the induction results of Agrobacterium rhizogenes selected from Ar1193 and ArA 4. A is the growth condition of stylosanthes guianensis hairy roots induced by Ar1193 agrobacterium rhizogenes; b is the growth condition of the hairy roots of the stylosanthes guianensis induced by the ArA4 agrobacterium rhizogenes; c is the detection result of the hairy roots of the stylosanthes guianensis induced by the selected Ar1193 agrobacterium rhizogenes; d is a detection result of selecting ArA4 rhizogenes agrobacterium rhizogenes to induce the hairy roots of the guide pillar flowers and plants.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
The variety of the stylosanthes guianensis is RY2 and is provided by the crop research institute of tropical academy of agricultural sciences in Hainan.
Example 1
Method and device
1. Media preparation
(1) The germination medium (1 × MS medium) consisted of: macroelements: 1.65 g.L-1NH4NO3、1.9g·L- 1KNO3、0.44g·L-1CaCl2·2H2O、0.37g·L-1MgSO4·7H2O、0.17g·L-1KH2PO4(ii) a Trace elements: 0.83 mg. L-1KI、6.2mg L-1H3BO3、22.3mg·L-1MnSO4·4H2O、10.6mg·L-1ZnSO4.7H2O、0.25mg·L-1Na2MoO4·2H2O、0.025mg·L-1CuSO4·5H2O、0.025mg·L-1CoCl2·6H2O、37.3mg·L-1Fe-EDTA; 1% sucrose; 0.3% vegetable gum. Sterilizing the germination medium at 121 ℃ for 20 minutes; after cooling to 60 ℃, the medium was poured into a square petri dish with a diameter of 13 × 13 cm.
(2) The preparation method of the agrobacterium rhizogenes culture medium comprises the following steps: YEP medium composition is: 10 g.L-1Tryptone, 10 g.L-1Yeast extract, 5 g.L-1NaCl，15g·L-1Agar (liquid medium does not need to be added). After preparation, sterilizing the mixture for 20 minutes at 121 ℃ in a sterilizing pot, then cooling the mixture to 60 ℃, and pouring the cooled mixture into a round glass bacteria culture dish for solidification for later use. Liquid YEP medium was stored at 4 ℃ until use.
(3) The composition of the co-culture solution for the induction of the hairy roots of stylosanthes guianensis is as follows: 0.4125 g.L-1NH4NO3、0.48g·L-1KNO3、0.11g·L-1CaCl2·2H2O、0.0925g·L-1MgSO4·7H2O、0.0425g·L-1KH2PO4、0.2075mg·L-1KI、1.54mg L-1H3BO3、5.56mg·L-1MnSO4·4H2O、2.65mg·L-1ZnSO4.7H2O、0.0625mg·L- 1Na2MoO4·2H2O、0.00625mg·L-1CuSO4·5H2O、0.00625mg·L-1CoCl2·6H2O、9.325mg·L-1Fe-EDTA、25mg·L-1Inositol, 0.5 mg.L-1Glycine; 0.5% sucrose. Sterilizing the culture solution at 121 deg.C for 20 min; and cooling for later use. Filter paper was placed in a sterile circular petri dish with a diameter of 11cm, and the culture solution was poured into the petri dish.
(4) The composition of the degerming culture medium is as follows: macroelement 1.65 g.L-1NH4NO3、1.9g·L-1KNO3、0.44g·L- 1CaCl2·2H2O、0.37g·L-1MgSO4·7H2O、0.17g·L-1KH2PO4(ii) a Trace elements: 0.83 mg. L-1KI、6.2mg L-1H3BO3、22.3mg·L-1MnSO4·4H2O、10.6mg·L-1ZnSO4.7H2O、0.25mg·L-1Na2MoO4·2H2O、0.025mg·L-1CuSO4·5H2O、0.025mg·L-1CoCl2·6H2O、37.3mg·L-1Fe-EDTA; 1% sucrose; 0.3% vegetable gum, 5g/LCarb
2. The operating steps of the method for inducing the hairy roots of stylosanthes guianensis are shown in figure 1, and the method specifically comprises the following steps:
(1) sterilizing and accelerating germination of stylosanthes guianensis seeds: removing episperm from Stylosanthes guianensis seed, placing into 1.5mL centrifuge tube to 0.5mL, adding 1mL sterile water, standing in 98 deg.C air bath for 3 min to break dormancy, removing sterile water with pipettor, addingSoaking seeds in 75% alcohol, intermittently shaking to thoroughly disinfect the seeds, and removing the alcohol after 1 minute; adding sterile water for cleaning for 3 times, removing the sterile water, adding 10% (v/v) sodium hypochlorite, intermittently shaking, removing the sodium hypochlorite after 5 minutes, adding sterile water for washing, and repeating the sterile water washing step for 3 times until the sodium hypochlorite is thoroughly washed. Then carefully sowing the sterilized plant seeds on the surface of the germination culture medium by using sterile toothpicks, covering the surface of the germination culture medium, sealing the surface of the germination culture medium along the edges by using a breathable medical adhesive tape, transferring the germination culture medium to an incubator, and culturing the germination culture medium by illumination. The conditions in the incubator were: the temperature in the day and night is 23 ℃, and the illumination intensity is 120 mu mol.m-2·S-1The light exposure time was 16 hours per day and the relative humidity was 60%.
(2) And (3) selecting and culturing agrobacterium rhizogenes: the Agrobacterium rhizogenes strain was MSU440 (supplied by Shanghai Weidi Biotechnology Co., Ltd.) harboring pTF101s binary vector plasmid. A small amount of MSU440 bacterial liquid is picked up by a sterile toothpick and streaked in a solid YEP culture medium. After the monoclonal colony grows out, picking the single colony from the YEP solid plate, inoculating the single colony into 0.5mL of YEP liquid culture medium, shaking and culturing on a constant temperature shaking table at 28 ℃ and at the rotating speed of 180r/min overnight to obtain seed bacterial liquid. And (3) sucking 200 mu L of the seed bacterial liquid, inoculating the seed bacterial liquid into 5mL of YEP liquid culture medium, shaking the seed bacterial liquid on a constant temperature shaking bed at the temperature of 28 ℃, and carrying out shaking culture at the rotating speed of 180r/min until the OD is 0.5-0.6.
(3) Infection of stylosanthes guianensis seedlings: taking out the seedlings of the stylosanthes guianensis which germinate for two days from the culture medium by using sterile tweezers, putting the seedlings into a sterilized culture dish, dipping the agrobacterium rhizogenes liquid by using a No. 11 sterile scalpel, and cutting off hypocotyls of the stylosanthes guianensis at a position 1mm below leaf nodes of the stylosanthes guianensis. Pouring a proper amount of agrobacterium rhizogenes bacterial liquid into a culture dish, cutting two cotyledons in the bacterial liquid, removing buds, and soaking the cut cotyledons in the YEP bacterial liquid for 30 minutes to finish the infection process.
(4) Induction of stylosanthes guianensis hairy roots: placing the sterilized filter paper into a circular culture dish with the diameter of 11cm, adding sterilized water to wet the filter paper to saturate the filter paper, transferring the stylosanthes guianensis plant exosome infected for 30 minutes into the culture dish, covering a cover, sealing the edge by using a non-breathable preservative film, and transferring the stylosanthes guianensis plant exosome into a light culture box to culture for 5 days; the conditions in the incubator were: the temperature in the day and at night was 25 ℃, the illumination intensity was 39060lx, the illumination time was 16 hours per day, and the relative humidity was 50%. And transferring the externally planted stylosanthes guianensis body after co-culture into a culture dish containing a co-culture solution, covering a cover, sealing the edge by using a non-breathable preservative film, transferring the externally planted stylosanthes guianensis body into an illumination incubator at the temperature of 23 ℃, wherein the illumination intensity is 39000-39100 lx, and co-culturing for about 14 days to obtain the hairy roots of stylosanthes guianensis with the length of about 2 cm.
(5) And (3) carrying out bacteria removal and propagation on the hairy roots of stylosanthes guianensis: after the hairy roots grow to be more than 2cm, the hairy root transformants are washed three times by sterile water (5g/L Carb) containing antibiotics, transferred to a sterile medium containing 5g/L Carb, subcultured 1 time every 7 days until the agrobacterium is completely killed, and finally transferred to 1/2 XMS medium to propagate the hairy roots.
3. Detection of transgenic hairy roots: about 0.05g of the secondary hairy roots were put into a 2mL centrifuge tube, and DNA of the sample was extracted by the CTAB method, followed by 2 × Rapid Master Mix detection of the herbicide reporter gene. The detection primers are as follows: Bar-F: CAACCACTACATCGAGACAAGCA, Bar-R: TCATCAGATCTCGGTGACGGG; a20 μ L PCR amplification system comprised: 10 μ L of Rapid Master Mix, 0.5 μ L of forward primer, 0.5 μ L of reverse primer, 1 μ L of DNA, 8 μ L of ddH2And O. Reaction procedure: pre-denaturation at 98 deg.C for 5min, 35 cycles of reaction (denaturation at 98 deg.C for 30s, annealing at 59 deg.C for 30s, extension at 72 deg.C for 1min), maintaining at 72 deg.C for 10min, and storing the amplification product at 16 deg.C after amplification.
Second, result in
And (3) selecting 24 hairy roots of 10 explants for detection, wherein the result is shown in figure 2, and 14 hairy roots are detected to have strips in the electrophoresis result and are positive hairy roots, so that the positive rate of the hairy roots is 58.3%.
Comparative example
Ar1193 and ArA4 Agrobacterium rhizogenes were selected, the other culture conditions were the same as in example 1, the induction of stylosanthes guianensis hairy roots is shown in FIGS. 3A-B, and the induction rates of Ar1193 and ArA4 were 23.4% and 28.5%, respectively; through DNA detection, the induction result of Ar1193 Agrobacterium rhizogenes is selected as shown in figure 3C, the positive rate of the hairy root is 0%, the induction result of ArA4 Agrobacterium rhizogenes is selected as shown in figure 3D, and the positive rate of the hairy root is 8.3%.
Claims (8)
1. A stylosanthes guianensis hairy root induction method is characterized by comprising the following steps:
s1, selection and culture of agrobacterium rhizogenes: activating and culturing an agrobacterium rhizogenes strain MSU440 into an agrobacterium rhizogenes liquid for later use;
s2, obtaining a stylosanthes guianensis explant: cutting the cotyledon node and/or cotyledon of the germinated stylosanthes guianensis seedling to be an explant;
s3, infecting stylosanthes guianensis explants: co-culturing the stylosanthes guianensis explant in the step S2 with the agrobacterium rhizogenes liquid in the step S1;
s4, inducing the styloid roots of stylosanthes guianensis: co-culturing the explant of the stylosanthes guianensis infected in the step S3 in sterile water for 4-6 days, transferring the explant to sterile filter paper soaked in the co-culture solution for co-culturing for 12-15 days, and preliminarily obtaining hairy roots at the cut;
s5, hairy root bacteria-removing culture and propagation: and (3) after the hairy roots grow to be more than 2cm, washing the hairy root transformants with sterile water containing antibiotics for three times, transferring the hairy root transformants into an MS culture medium containing antibiotics, subculturing for a plurality of times until the agrobacterium rhizogenes is completely killed, and finally transferring the hairy roots into 1/2MS culture medium to propagate the hairy roots.
2. The method according to claim 1, wherein the Agrobacterium rhizogenes solution obtained in step S1 is obtained by extracting 200-300 μ L of activated Agrobacterium rhizogenes strain MSU440 seed solution, inoculating into 5-10 mL YEP liquid medium, and shake culturing at 26-28 ℃ and 170-190 r/min until OD is 0.5-0.6.
3. The method according to claim 1, wherein the germination of step S2 is: sterilizing and disinfecting the stylosanthes guianensis seeds after dormancy breaking, and placing the stylosanthes guianensis seeds in a germination culture medium for culturing and germinating; the culture temperature is 22-24 ℃, and the illumination intensity is 110-130 mu mol.m-2·S-1The illumination time is 15-17 hours per day, and the relative humidity is 55-65%.
4. The method of claim 1, wherein the stylosanthes guianensis explant is a stylosanthes guianensis seedling cotyledon with a 1mm hypocotyl that germinates for 2 days.
5. The method according to claim 1, wherein the co-cultivation liquid of step S4 is composed of: 0.4125 g.L-1NH4NO3、0.48g·L-1KNO3、0.11g·L-1CaCl2·2H2O、0.0925g·L-1MgSO4·7H2O、0.0425g·L- 1KH2PO4、0.2075mg·L-1KI、1.54mg L-1H3BO3、5.56mg·L-1MnSO4·4H2O、2.65mg·L- 1ZnSO4.7H2O、0.0625mg·L-1Na2MoO4·2H2O、0.00625mg·L-1CuSO4·5H2O、0.00625mg·L- 1CoCl2·6H2O、9.325mg·L-1Fe-EDTA、25mg·L-1Inositol, 0.5 mg.L-1Glycine; 0.5% sucrose.
6. The method according to claim 1, wherein the step S4 is carried out by culturing in sterile water at 24-26 ℃, with an illumination intensity of 39000-39100 lx, an illumination time of 15-17 hours per day and a relative humidity of 45-55% for 4-6 co-culturing; the temperature of the stylosanthes guianensis explant and the co-culture solution are 22-24 ℃, the illumination intensity is 39000-39100 lx, and the illumination co-culture lasts for 12-15 days.
7. The method according to claim 1, wherein the antibiotic in the antibiotic-containing sterile water and the antibiotic-containing MS medium in step S5 is carbenicillin at a concentration of 4-6 g/L.
8. The method of claim 1, wherein the Stylosanthes guianensis variety RY 2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110879039.5A CN113604498B (en) | 2021-08-02 | 2021-08-02 | Method for inducing hairy roots of stylosanthes guianensis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110879039.5A CN113604498B (en) | 2021-08-02 | 2021-08-02 | Method for inducing hairy roots of stylosanthes guianensis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113604498A true CN113604498A (en) | 2021-11-05 |
| CN113604498B CN113604498B (en) | 2024-04-12 |
Family
ID=78306382
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110879039.5A Active CN113604498B (en) | 2021-08-02 | 2021-08-02 | Method for inducing hairy roots of stylosanthes guianensis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113604498B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114657206A (en) * | 2022-03-17 | 2022-06-24 | 中国热带农业科学院热带作物品种资源研究所 | Genetic transformation method of stylosanthes guianensis |
| CN117925696A (en) * | 2024-01-25 | 2024-04-26 | 中国热带农业科学院热带作物品种资源研究所 | Method for inducing transgenic hairy roots by using stylosanthes guianensis and method for preparing protoplast |
| CN117925696B (en) * | 2024-01-25 | 2024-07-09 | 中国热带农业科学院热带作物品种资源研究所 | Method for inducing transgenic hairy roots by using stylosanthes guianensis and method for preparing protoplast |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102827756A (en) * | 2012-06-25 | 2012-12-19 | 华南农业大学 | Agrobacterium local invasion method applied in soybean hypocotyl explant transformation system, and device thereof |
| CN106106185A (en) * | 2016-08-17 | 2016-11-16 | 桂林伯林生物技术有限公司 | A kind of Cyclocarya paliurus Iljinskaja rooting of vitro seedling culture medium and root culture method |
| CN108048474A (en) * | 2017-11-10 | 2018-05-18 | 华南农业大学 | A kind of acid phosphatase protein gene GmPAP1-like and its application |
| CN109511553A (en) * | 2019-01-24 | 2019-03-26 | 赵静 | A kind of coptis hairy highly effective revulsion induction method |
| CN111269931A (en) * | 2019-12-31 | 2020-06-12 | 贵州大学 | Agrobacterium rhizogenes-mediated flower tobacco hairy root induction transformation method |
| CN112119912A (en) * | 2020-08-28 | 2020-12-25 | 中国农业科学院油料作物研究所 | Method for quickly and efficiently establishing genetic transformation system of sesame hairy roots |
-
2021
- 2021-08-02 CN CN202110879039.5A patent/CN113604498B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102827756A (en) * | 2012-06-25 | 2012-12-19 | 华南农业大学 | Agrobacterium local invasion method applied in soybean hypocotyl explant transformation system, and device thereof |
| CN106106185A (en) * | 2016-08-17 | 2016-11-16 | 桂林伯林生物技术有限公司 | A kind of Cyclocarya paliurus Iljinskaja rooting of vitro seedling culture medium and root culture method |
| CN108048474A (en) * | 2017-11-10 | 2018-05-18 | 华南农业大学 | A kind of acid phosphatase protein gene GmPAP1-like and its application |
| CN109511553A (en) * | 2019-01-24 | 2019-03-26 | 赵静 | A kind of coptis hairy highly effective revulsion induction method |
| CN111269931A (en) * | 2019-12-31 | 2020-06-12 | 贵州大学 | Agrobacterium rhizogenes-mediated flower tobacco hairy root induction transformation method |
| CN112119912A (en) * | 2020-08-28 | 2020-12-25 | 中国农业科学院油料作物研究所 | Method for quickly and efficiently establishing genetic transformation system of sesame hairy roots |
Non-Patent Citations (5)
| Title |
|---|
| J M MANNERS ET AL.: "Efficient transformation with regeneration of the tropical pasture legumeStylosanthes humilis usingAgrobacterium rhizogenes and a Ti plasmid-binary vector system", 《PLANT CELL REP.》, vol. 8, no. 6, pages 341 * |
| 蒋昌顺;: "柱花草的研究进展", 热带作物学报, no. 04, pages 1 - 4 * |
| 陈彩虹: "根癌农杆菌介导柱花草遗传转化体系的优化", 《中国优秀硕士学位论文全文数据库（电子期刊）》, pages 11 * |
| 陈彩虹: "根癌农杆菌介导柱花草遗传转化体系的优化", 《海南大学》, no. 01 * |
| 陈燕,李聪,苏加楷,黄文惠,吕德杨,赵广宇: "发根农杆菌介导的百脉根的基因转化", 草地学报, no. 02, pages 116 - 120 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114657206A (en) * | 2022-03-17 | 2022-06-24 | 中国热带农业科学院热带作物品种资源研究所 | Genetic transformation method of stylosanthes guianensis |
| CN114657206B (en) * | 2022-03-17 | 2024-03-22 | 中国热带农业科学院热带作物品种资源研究所 | Genetic transformation method of stylosanthes guianensis |
| CN117925696A (en) * | 2024-01-25 | 2024-04-26 | 中国热带农业科学院热带作物品种资源研究所 | Method for inducing transgenic hairy roots by using stylosanthes guianensis and method for preparing protoplast |
| CN117925696B (en) * | 2024-01-25 | 2024-07-09 | 中国热带农业科学院热带作物品种资源研究所 | Method for inducing transgenic hairy roots by using stylosanthes guianensis and method for preparing protoplast |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113604498B (en) | 2024-04-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111454983A (en) | Preparation and transformation method of papaya callus agrobacterium transformation receptor | |
| CN112335549A (en) | Method for obtaining larch regeneration plant through tissue in-vitro culture | |
| CN112544440A (en) | Method for cultivating new transgenic or gene-edited seedlings of root tuber plants | |
| CN113604498B (en) | Method for inducing hairy roots of stylosanthes guianensis | |
| CN113265421B (en) | Method for establishing agrobacterium-mediated shortstem ephedra stem callus transgenic system | |
| CN106811482B (en) | Genetic transformation method for directly introducing exogenous gene by pansy seed soaking method | |
| CN105505991B (en) | A kind of quick transgenic method of longan | |
| CN111500620A (en) | Preparation and transformation method of papaya hypocotyl agrobacterium transformation receptor | |
| CN113528534B (en) | Application of GhMYB44 gene in differentiation and development of cotton callus | |
| CN113234750B (en) | Method for quickly obtaining transgenic plant taking isatis tinctoria transgenic root as explant | |
| CN113973658B (en) | Efficient genetic transformation and plant regeneration method for capsicum | |
| JP4228044B2 (en) | Redifferentiated plant and transformed plant of Shiba spp. | |
| JP2008259497A (en) | Method for creating transformant of domestic variety of soybean through agrobacterium and method for acquiring seed of current generation and progeny of transformant in short period of time | |
| CN113025645A (en) | Method for obtaining gypsophila paniculata transgenic plant by taking callus as receptor | |
| CN112931200B (en) | Tissue culture method using dianthus chinensis cotyledon and application of tissue culture method in dianthus chinensis genetic transformation | |
| Crane et al. | Medicago truncatula transformation using root explants | |
| KR100375674B1 (en) | A method of regenerating and transforming chinese cabbage(Brassica campestris ssp. pekinensis) using hypocotyl segments | |
| Duhoux et al. | Casuarina and Allocasuarina species | |
| CN114517202B (en) | Application of aquaporin gene SlPIP, 2 in improving resistance of tomatoes to facility continuous cropping soil | |
| AU2005330877B2 (en) | Method of constructing transgenic ice plant | |
| WO2007064037A1 (en) | Method for transformation of plant belonging to family araceae | |
| CN117660521A (en) | Transformation method for rapidly and efficiently inducing transgenic hairy roots of cotton | |
| CN111100871A (en) | Method for reducing browning and improving genetic transformation rate of agrobacterium-mediated castor | |
| CN112725376A (en) | High-efficiency genetic transformation method for agrobacterium-mediated buckwheat | |
| Padole et al. | In planta transformation studies in Gossypium hirsutum (L.) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |