Poison ivy (Toxicodendron radicans) is best known for causing exasperating allergenic delayed‐contact dermatitis symptoms that last for weeks on persons who have contacted the plant. 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, Agrobacterium rhizogenes was used to generate hormone‐independent poison ivy hairy root cultures. Optimal conditions for hairy root formation were skotomorphic poison ivy hypocotyls prick‐inoculated with A. rhizogenes, and preferential propagation of cultures with an atypical clumpy hairy root growth habit. The origin of the poison ivy accession used for A. rhizogenes prick‐inoculation did not affect the initial formation of calli/hairy root primordia, but rather significantly influenced the establishment of long‐term hormone‐independent hairy root growth. A. rhizogenes harboring a recombinant T‐DNA binary plasmid with an intron‐containing Firefly Luciferase gene produced stable transgenic hairy root lines expressing luciferase activity at high frequency. Poison ivy hairy root lines produced significantly lower steady‐state urushiol levels relative to wild‐type 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‐type roots as well. This study establishes a transformation hairy root regeneration protocol for poison ivy that can serve as a platform for future reverse‐genetic studies of urushiol biosynthesis in poison ivy hairy roots.

中文翻译：

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毒藤毛状根培养物能够形成适合漆酚代谢详细研究的稳定转化系统。


毒藤（ Toxicodendron radicans ）因引起令人恼火的过敏性迟发性接触性皮炎症状而闻名，接触过这种植物的人会持续数周。漆酚是由毒葛产生的烷基儿茶酚，是引起这种皮炎的原因。虽然漆酚的化学结构众所周知，但负责其生物合成的代谢中间体和基因尚未经过实验验证。毒葛中漆酚生物合成的分子遗传特征需要稳定的遗传转化和随后保留合成漆酚能力的器官再生。为此，使用发根农杆菌来产生不依赖激素的毒葛毛状根培养物。毛状根形成的最佳条件是用发根农杆菌点刺暗形毒葛下胚轴，并优先繁殖具有非典型丛生毛状根生长习性的培养物。用于发根农杆菌点刺接种的毒藤材料的来源并不影响愈伤组织/毛状根原基的初始形成，而是显着影响长期不依赖激素的毛状根生长的建立。带有含有内含子的萤火虫荧光素酶基因的重组T-DNA二元质粒的发根农杆菌产生了稳定的转基因毛状根系，该系以高频率表达荧光素酶活性。与野生型根相比，毒葛毛状根系产生的稳态漆酚水平明显较低，但漆酚水平高于漆酚水平不可检测的毒葛未分化愈伤组织系，这表明漆酚生物合成需要完整的毒葛器官。 毒藤毛状根系中较低的漆酚水平有助于首次在毛状根中以及随后在野生型根中首次鉴定漆树酸代谢物。本研究建立了毒葛毛状根再生的转化方案，可以作为未来毒葛毛状根漆酚生物合成反向遗传学研究的平台。