Germinating and Culturing Axenic Poison Ivy Seedlings

Urushiols are the chemical constituents responsible for causing the characteristic skin rash resulting from contact with poison ivy [Toxicodendron radicans subsp. radicans (L.) Kuntze] plant tissue. Future detailed physiological and molecular studies of T. radicans urushiol metabolism will require the production and cultivation of axenic T. radicans plants in controlled environments. To this end, the present study focused on treatments to enhance germination and reduce microbial contamination to obtain axenic T. radicans seedlings. Toxicodendron radicans drupes treated singularly with water, bleach, cold, or gibberellic acid showed very low germination frequencies. In contrast, concentrated sulfuric acid strongly promoted seedling germination by removing exocarp, mesocarp, and causing pitting of the brachysclereid and osteosclereid layers of the endocarp. Most T. radicans drupes harbored significant amounts of fungal and bacterial contaminants. Although the serial mechanical scarification, sulfuric acid, and bleach treatments promoted seedling germination, this serial treatment regime was not adequate to render the majority of drupes microbe-free. Nevertheless, ’25% of treated T. radicans drupes were axenic, and these needed to be promptly separated from adjacent fungal-contaminated drupes to avoid cross-contamination. The isolated axenic T. radicans drupes germinated at high frequency producing viable seedlings that grew well in sterile plant culture conditions. Poison ivy is best known for its ability to cause irritating skin rashes called Rhus dermatitis. Poison ivy belongs to the family Anacardiaceae, which includes other species producing sap capable of causing skin reactions, including poison oak (formerly Rhus toxicodendron diversilobum), poison sumac (formerly Rhus toxicodendron vernix), mango (Mangifera indica), cashew (Anacardium occidentale), and the Asian lac tree (Rhus verniciflua). Gillis proposed a systematic revision of poison ivy, poison oak, and poison sumac from the genera Rhus to Toxicodendron (Toxicodendron radicans, Toxicodendron diversilobum, and Toxicodendron vernix, respectively) (Gillis, 1971). Despite the moniker ‘‘poison’’ ivy, the manifested dermatitis is an immunologically based allergic reaction, delayed contact hypersensitivity, not an acute toxicity or poisoning (Kurtz and Dawson, 1971). The natural product responsible for inducing the delayed contact dermatitis is generically called urushiol. Urushiol refers to a number of pentadecylcatechol or heptadecylcatechol congeners with varying degrees of unsaturation ranging from one to three double bonds (Symes and Dawson, 1953, 1954). Urushiol triolefin congeners correlate with increased severity of contact dermatitis symptoms compared with those with less unsaturation (Johnson et al., 1972). The principal urushiol congeners in T. radicans are the pentadecylcatechols. Although the chemical composition of T. radicans urushiol and clinical immunology of the delayed contact dermatitis is well documented, urushiol physiology and metabolism in T. radicans plants are poorly understood. Urushiol levels and composition vary in poison ivy plants. In one report, young leaves, fresh young stems, fruits, and bark showed high urushiol levels, and the triolefin congener comprised over half of the total urushiols present (Craig et al., 1978). A different study examining leaves of different ages also showed high urushiol levels in young leaves with lower urushiol levels in the oldest leaves (Baer et al., 1980). In the latter study, the diolefin was the most abundant congener, whereas the triolefin was the least abundant. Both reports used tissues obtained from unmanaged T. radicans plants in which genetic, abiotic, and biotic factors were neither determined nor controlled. From these two studies it is clear that urushiol composition and levels are not determinate traits in poison ivy. Therefore, similar to other defensive plant secondary metabolites, T. radicans urushiol levels and composition are likely to change in response to developmental, environmental, and biotic factors. As a prerequisite for future detailed urushiol physiological and metabolism studies, it will be necessary to grow T. radicans plants under well-controlled environmental conditions. Axenic plantlets grown on synthetic media provide optimal experimental control over most abiotic and biotic parameters. However, because T. radicans has no economic value other than being a noxious invasive plant, there is rather limited knowledge about T. radicans germination patterns. Two prior studies suggest that T. radicans drupes require scarification to initiate seedling germination. One study focused on T. radicans seed dispersal by birds and squirrels demonstrated that sandpaper scarification significantly increased seedling germination frequencies relative to untreated drupes (Penner et al., 1999). A different study oriented on T. radicans interactions with host tree species used a combination of physical (pounding) and chemical (sulfuric acid) treatments to obtain adequate seedling germination frequencies to support studies into the effects of host tree allelochemical effects on T. radicans seedling germination and growth (Talley et al., 1996). These limited T. radicans findings are consistent with more extensive seed germination studies of closely related Rhus species (Anacardiaceae). Untreated drupes from five Rhus species (Rhus glabra, R. typhina, R. virens, R. aromatica, and R. trilobata) show very low seedling germination rates during permissive germination conditions (Li et al., 1999c). However, drupes from all five species showed significantly increased germination (albeit to differing degrees) after sulfuric acid scarification. The five Rhus species differed in whether the embryo dormancy was enforced by physiological or physical mechanisms. Only R. aromatica showed physiological dormancy that was broken by gibberellic acid treatment (Li et al., 1999b). In contrast, treatments that disrupted the physical integrity of the endocarp were sufficient to induce seedling germination in the other four Rhus species, indicating physically enforced seed dormancy (Li et al., 1999c). In the case of R. glabra, physical seed dormancy is maintained by the water-impermeable endocarp tissue, in particular the outermost brachysclereid and internally proximal osteosclereid cell layers (Li et al., 1999a, 1999c). The waterimpermeable brachysclereid and osteosclereid layers are sensitive to disruption by acid treatment, thereby allowing water to penetrate the underlying macrosclereid layer resulting in embryo imbibition and dormancy release. Given our interest to investigate urushiol metabolism and physiology during sterile tissue culture conditions in the future, it is currently necessary to develop foundational methods to germinate and culture axenic T. radicans seedlings. To this end, the primary objective of the present study was to identify physical, chemical, and cultural treatments suitable for producing axenic T. radicans seedlings cultured on synthetic media. Materials and Methods Toxicodendron radicans drupe collection. Two lianas of T. radicans subsp. radicans were the source of drupes used in this study. Drupes from the RoaCo-1 liana, located in Received for publication 10 Sept. 2013. Accepted for publication 16 Oct. 2013. J.G.J. is grateful to Roger Harris and Alex Niemiera for sharing their insights and enthusiasm for this nascent poison ivy research project. We also appreciate the helpful comments from R.H. and Richard Veilleux during the manuscript internal review. To whom reprint requests should be addressed; e-mail jelesko@vt.edu. HORTSCIENCE VOL. 48(12) DECEMBER 2013 1525 PROPAGATION AND TISSUE CULTURE