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Germinating and Culturing Axenic Poison Ivy Seedlings

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Germinating and Culturing Axenic Poison Ivy Seedlings PROPAGATION AND TISSUE CULTURE HORTSCIENCE 48(12):1525–1529. 2013. Axenic plantlets grown on synthetic media provide optimal experimental control over most abiotic and biotic parameters. However, Germinating and Culturing Axenic because T. radicans has no economic value other than being a noxious invasive plant, Poison Ivy Seedlings there is rather limited knowledge about 1 T. radicans germination patterns. Two prior Elise B. Benhase and John G. Jelesko studies suggest that T. radicans drupes re- Plant Pathology, Physiology, and Weed Science Department, Virginia Tech, quire scarification to initiate seedling germi- 548 Latham Hall, Blacksburg, VA, 24061-0390 nation. One study focused on T. radicans seed dispersal by birds and squirrels demon- Additional index words. dermatitis, drupe, endocarp, mesocarp, tissue culture, Toxicodendron strated that sandpaper scarification significantly radicans, urushiol increased seedling germination frequencies Abstract. Urushiols are the chemical constituents responsible for causing the character- relative to untreated drupes (Penner et al., istic skin rash resulting from contact with poison ivy [Toxicodendron radicans subsp. 1999). A different study oriented on T. radi- radicans (L.) Kuntze] plant tissue. Future detailed physiological and molecular studies cans interactions with host tree species used of T. radicans urushiol metabolism will require the production and cultivation of axenic a combination of physical (pounding) and T. radicans plants in controlled environments. To this end, the present study focused on chemical (sulfuric acid) treatments to obtain treatments to enhance germination and reduce microbial contamination to obtain axenic adequate seedling germination frequencies T. radicans seedlings. Toxicodendron radicans drupes treated singularly with water, to support studies into the effects of host tree bleach, cold, or gibberellic acid showed very low germination frequencies. In contrast, allelochemical effects on T. radicans seedling concentrated sulfuric acid strongly promoted seedling germination by removing exo- germination and growth (Talley et al., 1996). carp, mesocarp, and causing pitting of the brachysclereid and osteosclereid layers of the These limited T. radicans findings are endocarp. Most T. radicans drupes harbored significant amounts of fungal and bacterial consistent with more extensive seed germi- contaminants. Although the serial mechanical scarification, sulfuric acid, and bleach nation studies of closely related Rhus species treatments promoted seedling germination, this serial treatment regime was not (Anacardiaceae). Untreated drupes from five adequate to render the majority of drupes microbe-free. Nevertheless, ’25% of treated Rhus species (Rhus glabra, R. typhina, R. T. radicans drupes were axenic, and these needed to be promptly separated from adjacent virens, R. aromatica,andR. trilobata) show fungal-contaminated drupes to avoid cross-contamination. The isolated axenic T. very low seedling germination rates during radicans drupes germinated at high frequency producing viable seedlings that grew well permissive germination conditions (Li et al., in sterile plant culture conditions. 1999c). However, drupes from all five spe- cies showed significantly increased germina- tion (albeit to differing degrees) after sulfuric acid scarification. The five Rhus species dif- Poison ivy is best known for its ability to and Dawson, 1953, 1954). Urushiol triolefin fered in whether the embryo dormancy was cause irritating skin rashes called Rhus der- congeners correlate with increased severity enforced by physiological or physical mecha- matitis. Poison ivy belongs to the family of contact dermatitis symptoms compared with nisms. Only R. aromatica showed physiolog- Anacardiaceae, which includes other species those with less unsaturation (Johnson et al., ical dormancy that was broken by gibberellic producing sap capable of causing skin re- 1972). The principal urushiol congeners in acid treatment (Li et al., 1999b). In contrast, actions, including poison oak (formerly Rhus T. radicans are the pentadecylcatechols. Al- treatments that disrupted the physical integ- toxicodendron diversilobum), poison sumac though the chemical composition of T. rad- rity of the endocarp were sufficient to induce (formerly Rhus toxicodendron vernix), mango icans urushiol and clinical immunology of seedling germination in the other four Rhus (Mangifera indica), cashew (Anacardium occi- the delayed contact dermatitis is well docu- species, indicating physically enforced seed dentale),andtheAsianlactree(Rhus vernici- mented, urushiol physiology and metabolism dormancy (Li et al., 1999c). In the case of flua). Gillis proposed a systematic revision of in T. radicans plants are poorly understood. R. glabra, physical seed dormancy is main- poison ivy, poison oak, and poison sumac Urushiol levels and composition vary in tained by the water-impermeable endocarp from the genera Rhus to Toxicodendron (Tox- poison ivy plants. In one report, young leaves, tissue, in particular the outermost brachyscler- icodendron radicans, Toxicodendron diversilo- fresh young stems, fruits, and bark showed eid and internally proximal osteosclereid cell bum,andToxicodendron vernix, respectively) high urushiol levels, and the triolefin congener layers (Li et al., 1999a, 1999c). The water- (Gillis, 1971). Despite the moniker ‘‘poison’’ comprised over half of the total urushiols impermeable brachysclereid and osteoscler- ivy, the manifested dermatitis is an immuno- present (Craig et al., 1978). A different study eid layers are sensitive to disruption by acid logically based allergic reaction, delayed con- examining leaves of different ages also showed treatment, thereby allowing water to penetrate tact hypersensitivity, not an acute toxicity or high urushiol levels in young leaves with lower the underlying macrosclereid layer resulting in poisoning (Kurtz and Dawson, 1971). urushiol levels in the oldest leaves (Baer et al., embryo imbibition and dormancy release. The natural product responsible for induc- 1980). In the latter study, the diolefin was the Given our interest to investigate urushiol ing the delayed contact dermatitis is generically most abundant congener, whereas the triole- metabolism and physiology during sterile called urushiol. Urushiol refers to a number of fin was the least abundant. Both reports used tissue culture conditions in the future, it is pentadecylcatechol or heptadecylcatechol con- tissues obtained from unmanaged T. radicans currently necessary to develop foundational geners with varying degrees of unsaturation plants in which genetic, abiotic, and biotic methods to germinate and culture axenic ranging from one to three double bonds (Symes factors were neither determined nor controlled. T. radicans seedlings. To this end, the primary From these two studies it is clear that urushiol objective of the present study was to identify composition and levels are not determinate physical, chemical, and cultural treatments traits in poison ivy. Therefore, similar to suitable for producing axenic T. radicans seed- Received for publication 10 Sept. 2013. Accepted other defensive plant secondary metabolites, lings cultured on synthetic media. for publication 16 Oct. 2013. T. radicans urushiol levels and composition J.G.J. is grateful to Roger Harris and Alex Niemiera are likely to change in response to develop- for sharing their insights and enthusiasm for this Materials and Methods nascent poison ivy research project. We also appre- mental, environmental, and biotic factors. As ciate the helpful comments from R.H. and Richard a prerequisite for future detailed urushiol Toxicodendron radicans drupe collection. Veilleux during the manuscript internal review. physiological and metabolism studies, it will Two lianas of T. radicans subsp. radicans 1To whom reprint requests should be addressed; be necessary to grow T. radicans plants under were the source of drupes used in this study. e-mail [email protected]. well-controlled environmental conditions. Drupes from the RoaCo-1 liana, located in HORTSCIENCE VOL. 48(12) DECEMBER 2013 1525 Catawba, VA, at GPS coordinates latitude subjected to a 30-min 50% bleach treatment then subsequently treated with sterile water, 37°22#57.19$ N, longitude 80°6#43.07$ W, before plating with the goal of drupe surface cold (2 to 6 weeks at 4 °C), 1 mg·mL–1 were harvested in August of 2012. Drupes sterilization. The initial drupe treatment exper- gibberellic acid (GA), or SA treatments. from the Huckleberry-1 liana, located on the iments used 50 treated drupes on 100 · 15-mm One-way ANOVA results indicated that both Huckleberry Trail in Blacksburg, VA, at GPS petri plates containing 0.5 · MS basal salts solid replication and sandpaper scarification were coordinates latitude 37°12#7.29$ N, longidue media. After initial chemical treatments, the not significant factors (F = 0.45, P = 0.50; and 80°24#900$ W, were harvested on 1 Sept. seeds were stored at room temperature in the F = 0.75, P = 0.53, respectively), so these two 2012. The panicles were separated from the dark for 7 d and then transferred to a Percival parameters were removed from subsequent lianas, leaves removed, placed in black CU-36l4 growth chamber (Percival Scientific, ANOVA analyses. Fig. 1 illustrates T. radi- 113.5-L plastic bags in an outdoor shed for Perry, IA) set for constant 28 °C and 16 h light. cans seedling germination frequencies in 3 d, and then moved to an air-conditioned Seedling germination was scored 4 weeks post-
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