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Urushiols are alkylcatechols produced by poison ivy responsible for causing this dermatitis. While urushiol chemical structures are well known, the metabolic intermediates and genes responsible for their biosynthesis have not been experimentally validated. A molecular genetic characterization of urushiol biosynthesis in poison ivy will require stable genetic transformation and subsequent regeneration of organs that retain the capacity synthesize urushiol. To this end, <jats:italic>Agrobacterium rhizogenes<\/jats:italic> was used to generate hormone\u2010independent poison ivy hairy root cultures. Optimal conditions for hairy root formation were skotomorphic poison ivy hypocotyls prick\u2010inoculated with <jats:italic>A.\u00a0rhizogenes<\/jats:italic>, and preferential propagation of cultures with an atypical clumpy hairy root growth habit. The origin of the poison ivy accession used for <jats:italic>A.\u00a0rhizogenes<\/jats:italic> prick\u2010inoculation did not affect the initial formation of calli\/hairy root primordia, but rather significantly influenced the establishment of long\u2010term hormone\u2010independent hairy root growth. <jats:italic>A.\u00a0rhizogenes<\/jats:italic> harboring a recombinant T\u2010DNA binary plasmid with an intron\u2010containing Firefly Luciferase gene produced stable transgenic hairy root lines expressing luciferase activity at high frequency. Poison ivy hairy root lines produced significantly lower steady\u2010state urushiol levels relative to wild\u2010type roots, but higher urushiol levels than a poison ivy undifferentiated callus line with undetectable urushiol levels, suggesting that urushiol biosynthesis requires intact poison ivy organs. The lower urushiol levels in poison ivy hairy root lines facilitated the first identification of anacardic acid metabolites initially in hairy roots, and subsequently in wild\u2010type roots as well. This study establishes a transformation hairy root regeneration protocol for poison ivy that can serve as a platform for future reverse\u2010genetic studies of urushiol biosynthesis in poison ivy hairy roots.<\/jats:p>","DOI":"10.1002\/pld3.243","type":"journal-article","created":{"date-parts":[[2020,8,7]],"date-time":"2020-08-07T08:43:43Z","timestamp":1596789823000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Poison ivy hairy root cultures enable a stable transformation system suitable for detailed investigation of urushiol metabolism"],"prefix":"10.1002","volume":"4","author":[{"given":"Aneirin A.","family":"Lott","sequence":"first","affiliation":[{"name":"Plant Molecular and Cellular Biology University of Florida Gainesville FL USA"},{"name":"School of Plant and Environmental Science Virginia Tech Blacksburg VA USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Catherine P.","family":"Freed","sequence":"additional","affiliation":[{"name":"Biochemistry Department Virginia Tech Blacksburg VA USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Christopher C.","family":"Dickinson","sequence":"additional","affiliation":[{"name":"School of Plant and Environmental Science Virginia Tech Blacksburg VA USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Susan R.","family":"Whitehead","sequence":"additional","affiliation":[{"name":"Biological Science Department Virginia Tech Blacksburg VA USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Eva","family":"Collakova","sequence":"additional","affiliation":[{"name":"School of Plant and Environmental Science Virginia Tech Blacksburg VA USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"John G.","family":"Jelesko","sequence":"additional","affiliation":[{"name":"School of Plant and Environmental Science Virginia Tech Blacksburg VA USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2020,8,7]]},"reference":[{"key":"e_1_2_8_2_1","doi-asserted-by":"publisher","DOI":"10.3390\/molecules22050711"},{"key":"e_1_2_8_3_1","doi-asserted-by":"publisher","DOI":"10.1016\/0031-9422(80)85114-4"},{"key":"e_1_2_8_4_1","doi-asserted-by":"publisher","DOI":"10.1007\/s10725-016-0145-3"},{"key":"e_1_2_8_5_1","doi-asserted-by":"publisher","DOI":"10.21273\/HORTSCI.48.12.1525"},{"key":"e_1_2_8_6_1","doi-asserted-by":"publisher","DOI":"10.1007\/BF00037278"},{"key":"e_1_2_8_7_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0031-9422(00)88965-7"},{"key":"e_1_2_8_8_1","doi-asserted-by":"publisher","DOI":"10.1007\/BF02823136"},{"key":"e_1_2_8_9_1","doi-asserted-by":"publisher","DOI":"10.1055\/s-2001-12006"},{"key":"e_1_2_8_10_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00299-006-0164-z"},{"key":"e_1_2_8_11_1","doi-asserted-by":"publisher","DOI":"10.1038\/295432a0"},{"key":"e_1_2_8_12_1","doi-asserted-by":"publisher","DOI":"10.1007\/PL00008126"},{"key":"e_1_2_8_13_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0168-9452(98)00149-6"},{"key":"e_1_2_8_14_1","doi-asserted-by":"publisher","DOI":"10.1016\/0168-9452(92)90147-E"},{"key":"e_1_2_8_15_1","doi-asserted-by":"publisher","DOI":"10.1002\/jps.2600670411"},{"key":"e_1_2_8_16_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00299-007-0365-0"},{"key":"e_1_2_8_17_1","doi-asserted-by":"publisher","DOI":"10.1626\/jcs.64.650"},{"key":"e_1_2_8_18_1","doi-asserted-by":"publisher","DOI":"10.1007\/BF00036800"},{"key":"e_1_2_8_19_1","volume-title":"Medicinal natural products: A biosynthetic approach","author":"Dewick P. 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