A simple and efficient protocol for generating transgenic hairy roots using Agrobacterium rhizogenes

For decades, Agrobacterium rhizogenes (now Rhizobium rhizogenes), the causative agent of hairy root disease, has been harnessed as an interkingdom DNA delivery tool for generating transgenic hairy roots on a wide variety of plants. One of the strategies involves the construction of transconjugant R....

Full description

Saved in:
Bibliographic Details
Published in:PloS one 2023-11, Vol.18 (11), p.e0291680-e0291680
Main Authors: Ferguson, Shaun, Abel, Nikolaj B, Reid, Dugald, Madsen, Lene H, Luu, Thi-Bich, Andersen, Kasper R, Stougaard, Jens, Radutoiu, Simona
Format: Article
Language:English
Subjects:
Agrobacterium rhizogenes
Analysis
Beans
Biology and life sciences
Cloning
Cloning vectors
DNA
E coli
Engineering and Technology
Genes
Genetic aspects
Genetic engineering
Genetic transformation
Gram-negative bacteria
Hairy root
Health aspects
Lab Protocol
Legumes
Medicine and Health Sciences
Mimosaceae
Nitrogen
Nodules
Parenting
Physiology
Plant bacterial diseases
Plant diseases
Plasmids
Protocol
Research and analysis methods
Roots
Workloads
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
cited_by cdi_FETCH-LOGICAL-c670t-eaa7c85ed45d280def2b0f41b4f429b86c880db01cd10a8836f6ad7fdaacae403
cites cdi_FETCH-LOGICAL-c670t-eaa7c85ed45d280def2b0f41b4f429b86c880db01cd10a8836f6ad7fdaacae403
container_end_page e0291680
container_issue 11
container_start_page e0291680
container_title PloS one
container_volume 18
creator Ferguson, Shaun
Abel, Nikolaj B
Reid, Dugald
Madsen, Lene H
Luu, Thi-Bich
Andersen, Kasper R
Stougaard, Jens
Radutoiu, Simona
description For decades, Agrobacterium rhizogenes (now Rhizobium rhizogenes), the causative agent of hairy root disease, has been harnessed as an interkingdom DNA delivery tool for generating transgenic hairy roots on a wide variety of plants. One of the strategies involves the construction of transconjugant R. rhizogenes by transferring gene(s) of interest into previously constructed R. rhizogenes pBR322 acceptor strains; little has been done, however, to improve upon this system since its implementation. We developed a simplified method utilising bi-parental mating in conjunction with effective counterselection for generating R. rhizogenes transconjugants. Central to this was the construction of a new Modular Cloning (MoClo) compatible pBR322-derived integration vector (pIV101). Although this protocol remains limited to pBR322 acceptor strains, pIV101 facilitated an efficient construction of recombinant vectors, effective screening of transconjugants, and RP4-based mobilisation compatibility that enabled simplified conjugal transfer. Transconjugants from this system were tested on Lotus japonicus and found to be efficient for the transformation of transgenic hairy roots and supported infection of nodules by a rhizobia symbiont. The expedited protocol detailed herein substantially decreased both the time and labour for creating transconjugant R. rhizogenes for the subsequent transgenic hairy root transformation of Lotus, and it could readily be applied for the transformation of other plants.
doi_str_mv 10.1371/journal.pone.0291680
format article
fullrecord <record><control><sourceid>gale_plos_</sourceid><recordid>TN_cdi_plos_journals_3069192160</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><galeid>A771106690</galeid><doaj_id>oai_doaj_org_article_75ff61ac662f42ff9eeccfa9d8dd0b1f</doaj_id><sourcerecordid>A771106690</sourcerecordid><originalsourceid>FETCH-LOGICAL-c670t-eaa7c85ed45d280def2b0f41b4f429b86c880db01cd10a8836f6ad7fdaacae403</originalsourceid><addsrcrecordid>eNqNk1uL1TAQx4so7rr6DQQLgujDOSa9pM2THBYvBxYWvL2GaTJpc2ibY5KK66c39VTZyj5IHpLM_OY_mSGTJE8p2dK8oq8PdnIj9NujHXFLMk5ZTe4l55Tn2YZlJL9_63yWPPL-QEiZ14w9TM7yilNSMnaeqF3qzXDsMYVRpai1kQbHkB6dDVbaPtXWpS2O6CCYsU2Dg9HHu5FpB8bdpM7a4NPJz85d62wDMqAz05C6zvy0c6h_nDzQ0Ht8suwXyZd3bz9ffthcXb_fX-6uNpJVJGwQoJJ1iaooVVYThTpriC5oU-gi403NZB2tDaFSUQJ1nTPNQFVaAUjAguQXybOT7rG3XiwN8iInjFOeUTYT-xOhLBzE0ZkB3I2wYMRvg3WtABeM7FFUpdaMgmQsi-m15ohSauCqVoo0VEetN0u2qRlQydg2B_1KdO0ZTSda-11QwiiveBkVXi4Kzn6b0AcxGC-x72FEO3mR1XVZ5pzn88Of_4PeXd5CtRArMKO2MbGcRcWuqmhMzPhMbe-g4lI4GBm_kzbRvgp4tQqITMAfoYXJe7H_9PH_2euva_bFLbZD6EPnbT8FY0e_BosTKJ313qH-22VKxDwNf7oh5mkQyzTkvwBab_3_</addsrcrecordid><sourcetype>Open Website</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>3069192160</pqid></control><display><type>article</type><title>A simple and efficient protocol for generating transgenic hairy roots using Agrobacterium rhizogenes</title><source>Publicly Available Content Database</source><source>PubMed</source><creator>Ferguson, Shaun ; Abel, Nikolaj B ; Reid, Dugald ; Madsen, Lene H ; Luu, Thi-Bich ; Andersen, Kasper R ; Stougaard, Jens ; Radutoiu, Simona</creator><contributor>Kordrostami, Mojtaba</contributor><creatorcontrib>Ferguson, Shaun ; Abel, Nikolaj B ; Reid, Dugald ; Madsen, Lene H ; Luu, Thi-Bich ; Andersen, Kasper R ; Stougaard, Jens ; Radutoiu, Simona ; Kordrostami, Mojtaba</creatorcontrib><description>For decades, Agrobacterium rhizogenes (now Rhizobium rhizogenes), the causative agent of hairy root disease, has been harnessed as an interkingdom DNA delivery tool for generating transgenic hairy roots on a wide variety of plants. One of the strategies involves the construction of transconjugant R. rhizogenes by transferring gene(s) of interest into previously constructed R. rhizogenes pBR322 acceptor strains; little has been done, however, to improve upon this system since its implementation. We developed a simplified method utilising bi-parental mating in conjunction with effective counterselection for generating R. rhizogenes transconjugants. Central to this was the construction of a new Modular Cloning (MoClo) compatible pBR322-derived integration vector (pIV101). Although this protocol remains limited to pBR322 acceptor strains, pIV101 facilitated an efficient construction of recombinant vectors, effective screening of transconjugants, and RP4-based mobilisation compatibility that enabled simplified conjugal transfer. Transconjugants from this system were tested on Lotus japonicus and found to be efficient for the transformation of transgenic hairy roots and supported infection of nodules by a rhizobia symbiont. The expedited protocol detailed herein substantially decreased both the time and labour for creating transconjugant R. rhizogenes for the subsequent transgenic hairy root transformation of Lotus, and it could readily be applied for the transformation of other plants.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0291680</identifier><identifier>PMID: 37910566</identifier><language>eng</language><publisher>San Francisco: Public Library of Science</publisher><subject>Agrobacterium rhizogenes ; Analysis ; Beans ; Biology and life sciences ; Cloning ; Cloning vectors ; DNA ; E coli ; Engineering and Technology ; Genes ; Genetic aspects ; Genetic engineering ; Genetic transformation ; Gram-negative bacteria ; Hairy root ; Health aspects ; Lab Protocol ; Legumes ; Medicine and Health Sciences ; Mimosaceae ; Nitrogen ; Nodules ; Parenting ; Physiology ; Plant bacterial diseases ; Plant diseases ; Plasmids ; Protocol ; Research and analysis methods ; Roots ; Workloads</subject><ispartof>PloS one, 2023-11, Vol.18 (11), p.e0291680-e0291680</ispartof><rights>COPYRIGHT 2023 Public Library of Science</rights><rights>2023 Ferguson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2023 Ferguson et al 2023 Ferguson et al</rights><rights>2023 Ferguson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c670t-eaa7c85ed45d280def2b0f41b4f429b86c880db01cd10a8836f6ad7fdaacae403</citedby><cites>FETCH-LOGICAL-c670t-eaa7c85ed45d280def2b0f41b4f429b86c880db01cd10a8836f6ad7fdaacae403</cites><orcidid>0000-0002-8968-7501 ; 0000-0002-4415-8067 ; 0000-0002-1677-0769</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.proquest.com/docview/3069192160/fulltextPDF?pq-origsite=primo$$EPDF$$P50$$Gproquest$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.proquest.com/docview/3069192160?pq-origsite=primo$$EHTML$$P50$$Gproquest$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,885,25751,27922,27923,37010,37011,44588,53789,53791,74896</link.rule.ids></links><search><contributor>Kordrostami, Mojtaba</contributor><creatorcontrib>Ferguson, Shaun</creatorcontrib><creatorcontrib>Abel, Nikolaj B</creatorcontrib><creatorcontrib>Reid, Dugald</creatorcontrib><creatorcontrib>Madsen, Lene H</creatorcontrib><creatorcontrib>Luu, Thi-Bich</creatorcontrib><creatorcontrib>Andersen, Kasper R</creatorcontrib><creatorcontrib>Stougaard, Jens</creatorcontrib><creatorcontrib>Radutoiu, Simona</creatorcontrib><title>A simple and efficient protocol for generating transgenic hairy roots using Agrobacterium rhizogenes</title><title>PloS one</title><description>For decades, Agrobacterium rhizogenes (now Rhizobium rhizogenes), the causative agent of hairy root disease, has been harnessed as an interkingdom DNA delivery tool for generating transgenic hairy roots on a wide variety of plants. One of the strategies involves the construction of transconjugant R. rhizogenes by transferring gene(s) of interest into previously constructed R. rhizogenes pBR322 acceptor strains; little has been done, however, to improve upon this system since its implementation. We developed a simplified method utilising bi-parental mating in conjunction with effective counterselection for generating R. rhizogenes transconjugants. Central to this was the construction of a new Modular Cloning (MoClo) compatible pBR322-derived integration vector (pIV101). Although this protocol remains limited to pBR322 acceptor strains, pIV101 facilitated an efficient construction of recombinant vectors, effective screening of transconjugants, and RP4-based mobilisation compatibility that enabled simplified conjugal transfer. Transconjugants from this system were tested on Lotus japonicus and found to be efficient for the transformation of transgenic hairy roots and supported infection of nodules by a rhizobia symbiont. The expedited protocol detailed herein substantially decreased both the time and labour for creating transconjugant R. rhizogenes for the subsequent transgenic hairy root transformation of Lotus, and it could readily be applied for the transformation of other plants.</description><subject>Agrobacterium rhizogenes</subject><subject>Analysis</subject><subject>Beans</subject><subject>Biology and life sciences</subject><subject>Cloning</subject><subject>Cloning vectors</subject><subject>DNA</subject><subject>E coli</subject><subject>Engineering and Technology</subject><subject>Genes</subject><subject>Genetic aspects</subject><subject>Genetic engineering</subject><subject>Genetic transformation</subject><subject>Gram-negative bacteria</subject><subject>Hairy root</subject><subject>Health aspects</subject><subject>Lab Protocol</subject><subject>Legumes</subject><subject>Medicine and Health Sciences</subject><subject>Mimosaceae</subject><subject>Nitrogen</subject><subject>Nodules</subject><subject>Parenting</subject><subject>Physiology</subject><subject>Plant bacterial diseases</subject><subject>Plant diseases</subject><subject>Plasmids</subject><subject>Protocol</subject><subject>Research and analysis methods</subject><subject>Roots</subject><subject>Workloads</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2023</creationdate><recordtype>article</recordtype><sourceid>PIMPY</sourceid><sourceid>DOA</sourceid><recordid>eNqNk1uL1TAQx4so7rr6DQQLgujDOSa9pM2THBYvBxYWvL2GaTJpc2ibY5KK66c39VTZyj5IHpLM_OY_mSGTJE8p2dK8oq8PdnIj9NujHXFLMk5ZTe4l55Tn2YZlJL9_63yWPPL-QEiZ14w9TM7yilNSMnaeqF3qzXDsMYVRpai1kQbHkB6dDVbaPtXWpS2O6CCYsU2Dg9HHu5FpB8bdpM7a4NPJz85d62wDMqAz05C6zvy0c6h_nDzQ0Ht8suwXyZd3bz9ffthcXb_fX-6uNpJVJGwQoJJ1iaooVVYThTpriC5oU-gi403NZB2tDaFSUQJ1nTPNQFVaAUjAguQXybOT7rG3XiwN8iInjFOeUTYT-xOhLBzE0ZkB3I2wYMRvg3WtABeM7FFUpdaMgmQsi-m15ohSauCqVoo0VEetN0u2qRlQydg2B_1KdO0ZTSda-11QwiiveBkVXi4Kzn6b0AcxGC-x72FEO3mR1XVZ5pzn88Of_4PeXd5CtRArMKO2MbGcRcWuqmhMzPhMbe-g4lI4GBm_kzbRvgp4tQqITMAfoYXJe7H_9PH_2euva_bFLbZD6EPnbT8FY0e_BosTKJ313qH-22VKxDwNf7oh5mkQyzTkvwBab_3_</recordid><startdate>20231101</startdate><enddate>20231101</enddate><creator>Ferguson, Shaun</creator><creator>Abel, Nikolaj B</creator><creator>Reid, Dugald</creator><creator>Madsen, Lene H</creator><creator>Luu, Thi-Bich</creator><creator>Andersen, Kasper R</creator><creator>Stougaard, Jens</creator><creator>Radutoiu, Simona</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</general><scope>AAYXX</scope><scope>CITATION</scope><scope>IOV</scope><scope>ISR</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QO</scope><scope>7RV</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TG</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>KB0</scope><scope>KL.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>NAPCQ</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PATMY</scope><scope>PDBOC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope><orcidid>https://orcid.org/0000-0002-8968-7501</orcidid><orcidid>https://orcid.org/0000-0002-4415-8067</orcidid><orcidid>https://orcid.org/0000-0002-1677-0769</orcidid></search><sort><creationdate>20231101</creationdate><title>A simple and efficient protocol for generating transgenic hairy roots using Agrobacterium rhizogenes</title><author>Ferguson, Shaun ; Abel, Nikolaj B ; Reid, Dugald ; Madsen, Lene H ; Luu, Thi-Bich ; Andersen, Kasper R ; Stougaard, Jens ; Radutoiu, Simona</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c670t-eaa7c85ed45d280def2b0f41b4f429b86c880db01cd10a8836f6ad7fdaacae403</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2023</creationdate><topic>Agrobacterium rhizogenes</topic><topic>Analysis</topic><topic>Beans</topic><topic>Biology and life sciences</topic><topic>Cloning</topic><topic>Cloning vectors</topic><topic>DNA</topic><topic>E coli</topic><topic>Engineering and Technology</topic><topic>Genes</topic><topic>Genetic aspects</topic><topic>Genetic engineering</topic><topic>Genetic transformation</topic><topic>Gram-negative bacteria</topic><topic>Hairy root</topic><topic>Health aspects</topic><topic>Lab Protocol</topic><topic>Legumes</topic><topic>Medicine and Health Sciences</topic><topic>Mimosaceae</topic><topic>Nitrogen</topic><topic>Nodules</topic><topic>Parenting</topic><topic>Physiology</topic><topic>Plant bacterial diseases</topic><topic>Plant diseases</topic><topic>Plasmids</topic><topic>Protocol</topic><topic>Research and analysis methods</topic><topic>Roots</topic><topic>Workloads</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Ferguson, Shaun</creatorcontrib><creatorcontrib>Abel, Nikolaj B</creatorcontrib><creatorcontrib>Reid, Dugald</creatorcontrib><creatorcontrib>Madsen, Lene H</creatorcontrib><creatorcontrib>Luu, Thi-Bich</creatorcontrib><creatorcontrib>Andersen, Kasper R</creatorcontrib><creatorcontrib>Stougaard, Jens</creatorcontrib><creatorcontrib>Radutoiu, Simona</creatorcontrib><collection>CrossRef</collection><collection>Gale In Context: Opposing Viewpoints</collection><collection>Gale In Context: Science</collection><collection>ProQuest Central (Corporate)</collection><collection>Animal Behavior Abstracts</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Proquest Nursing &amp; Allied Health Source</collection><collection>Ecology Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Immunology Abstracts</collection><collection>Meteorological &amp; Geoastrophysical Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Agricultural Science Collection</collection><collection>Health &amp; Medical Collection (Proquest)</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Public Health Database (Proquest)</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Technology Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>Materials Science &amp; Engineering Collection</collection><collection>ProQuest Central (Alumni)</collection><collection>ProQuest One Sustainability</collection><collection>ProQuest Central UK/Ireland</collection><collection>Advanced Technologies &amp; Aerospace Database‎ (1962 - current)</collection><collection>ProQuest Agriculture &amp; Environmental Science Database</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Technology Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Materials Science Collection</collection><collection>ProQuest Central</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health &amp; Medical Complete (Alumni)</collection><collection>ProQuest Materials Science Database</collection><collection>Nursing &amp; Allied Health Database (Alumni Edition)</collection><collection>Meteorological &amp; Geoastrophysical Abstracts - Academic</collection><collection>ProQuest Engineering Collection</collection><collection>Biological Sciences</collection><collection>Agriculture Science Database</collection><collection>Health &amp; Medical Collection (Alumni Edition)</collection><collection>PML(ProQuest Medical Library)</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biological Science Database</collection><collection>ProQuest Engineering Database</collection><collection>Nursing &amp; Allied Health Premium</collection><collection>ProQuest Advanced Technologies &amp; Aerospace Database</collection><collection>ProQuest Advanced Technologies &amp; Aerospace Collection</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Environmental Science Database</collection><collection>Materials Science Collection</collection><collection>Publicly Available Content Database</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>Engineering collection</collection><collection>Environmental Science Collection</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Ferguson, Shaun</au><au>Abel, Nikolaj B</au><au>Reid, Dugald</au><au>Madsen, Lene H</au><au>Luu, Thi-Bich</au><au>Andersen, Kasper R</au><au>Stougaard, Jens</au><au>Radutoiu, Simona</au><au>Kordrostami, Mojtaba</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A simple and efficient protocol for generating transgenic hairy roots using Agrobacterium rhizogenes</atitle><jtitle>PloS one</jtitle><date>2023-11-01</date><risdate>2023</risdate><volume>18</volume><issue>11</issue><spage>e0291680</spage><epage>e0291680</epage><pages>e0291680-e0291680</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>For decades, Agrobacterium rhizogenes (now Rhizobium rhizogenes), the causative agent of hairy root disease, has been harnessed as an interkingdom DNA delivery tool for generating transgenic hairy roots on a wide variety of plants. One of the strategies involves the construction of transconjugant R. rhizogenes by transferring gene(s) of interest into previously constructed R. rhizogenes pBR322 acceptor strains; little has been done, however, to improve upon this system since its implementation. We developed a simplified method utilising bi-parental mating in conjunction with effective counterselection for generating R. rhizogenes transconjugants. Central to this was the construction of a new Modular Cloning (MoClo) compatible pBR322-derived integration vector (pIV101). Although this protocol remains limited to pBR322 acceptor strains, pIV101 facilitated an efficient construction of recombinant vectors, effective screening of transconjugants, and RP4-based mobilisation compatibility that enabled simplified conjugal transfer. Transconjugants from this system were tested on Lotus japonicus and found to be efficient for the transformation of transgenic hairy roots and supported infection of nodules by a rhizobia symbiont. The expedited protocol detailed herein substantially decreased both the time and labour for creating transconjugant R. rhizogenes for the subsequent transgenic hairy root transformation of Lotus, and it could readily be applied for the transformation of other plants.</abstract><cop>San Francisco</cop><pub>Public Library of Science</pub><pmid>37910566</pmid><doi>10.1371/journal.pone.0291680</doi><tpages>e0291680</tpages><orcidid>https://orcid.org/0000-0002-8968-7501</orcidid><orcidid>https://orcid.org/0000-0002-4415-8067</orcidid><orcidid>https://orcid.org/0000-0002-1677-0769</orcidid><oa>free_for_read</oa></addata></record>
fulltext fulltext
identifier ISSN: 1932-6203
ispartof PloS one, 2023-11, Vol.18 (11), p.e0291680-e0291680
issn 1932-6203
1932-6203
language eng
recordid cdi_plos_journals_3069192160
source Publicly Available Content Database; PubMed
subjects Agrobacterium rhizogenes
Analysis
Beans
Biology and life sciences
Cloning
Cloning vectors
DNA
E coli
Engineering and Technology
Genes
Genetic aspects
Genetic engineering
Genetic transformation
Gram-negative bacteria
Hairy root
Health aspects
Lab Protocol
Legumes
Medicine and Health Sciences
Mimosaceae
Nitrogen
Nodules
Parenting
Physiology
Plant bacterial diseases
Plant diseases
Plasmids
Protocol
Research and analysis methods
Roots
Workloads
title A simple and efficient protocol for generating transgenic hairy roots using Agrobacterium rhizogenes
url http://sfxeu10.hosted.exlibrisgroup.com/loughborough?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-13T17%3A06%3A53IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=A%20simple%20and%20efficient%20protocol%20for%20generating%20transgenic%20hairy%20roots%20using%20Agrobacterium%20rhizogenes&rft.jtitle=PloS%20one&rft.au=Ferguson,%20Shaun&rft.date=2023-11-01&rft.volume=18&rft.issue=11&rft.spage=e0291680&rft.epage=e0291680&rft.pages=e0291680-e0291680&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0291680&rft_dat=%3Cgale_plos_%3EA771106690%3C/gale_plos_%3E%3Cgrp_id%3Ecdi_FETCH-LOGICAL-c670t-eaa7c85ed45d280def2b0f41b4f429b86c880db01cd10a8836f6ad7fdaacae403%3C/grp_id%3E%3Coa%3E%3C/oa%3E%3Curl%3E%3C/url%3E&rft_id=info:oai/&rft_pqid=3069192160&rft_id=info:pmid/37910566&rft_galeid=A771106690&rfr_iscdi=true