HORTSCIENCE 49(2):194–200. 2014. species listed under the ESA, has outlined recovery criteria for Ruth’s golden aster that highlight the most critical data gaps and Propagation for the Conservation of research needs [U.S. Fish and Wildlife Ser- vice (USFWS), 1992]. The most recent re- Pityopsis ruthii, an Endangered Species view of the species status cites a continuing need for better ex situ conservation efforts from the Southeastern United States and additional research focused on restora- tion of Ruth’s golden aster in suitable habitat Phillip A. Wadl1 (USFWS, 2012). Department of Entomology and Plant Pathology, University of Tennessee, Previous ex situ conservation efforts for 2505 E.J. Chapman Drive, 370 Plant Biotechnology Building, Knoxville, Ruth’s golden aster have been focused en- tirely on long-term seed storage (USFWS, TN 37996 2012). Provided seeds are available and Timothy A. Rinehart germinate readily, seed-based methods are often the most efficient means for ex situ Thad Cochran Southern Horticulture Research Laboratory, U.S. Department conservation (Pence, 2011). However, wild- of Agriculture–Agricultural Research Service (USDA-ARS), 810 Highway 26 collected Ruth’s golden aster seeds often West, Poplarville, MS 39470 exhibit poor germination and produce seed- lings with low vigor (Clebsch and Sloan, Adam J. Dattilo 1993; Cruzan, 2001; Farmer, 1977; White, Biological Compliance, Tennessee Valley Authority, West Tower 11C-K, 400 1977). To overcome the limitations of prop- West Summit Hill Drive, Knoxville, TN 37902 agating solely with seed and to provide increased flexibility in ex situ conservation Mark Pistrang efforts, an in vitro propagation protocol was Cherokee National Forest, U.S. Department of Agriculture–Forest Service, developed (Wadl et al., 2011). Whereas Wadl 2800 North Ocoee Street, Cleveland, TN 37312 et al. (2011) demonstrated the capability to produce plants in vitro, further refinement Lisa M. Vito, Ryan Milstead, and Robert N. Trigiano of current techniques is needed to ensure Department of Entomology and Plant Pathology, University of Tennessee, reliable propagation. Although propagating Ruth’s golden aster from seed can be prob- 2505 E.J. Chapman Drive, 370 Plant Biotechnology Building, Knoxville, TN lematic, using seed has the advantage of 37996 producing genetically diverse propagules in- Additional index words. bonded fiber matrix, ex situ, in vitro, reintroduction, Ruth’s golden stead of clonal plants, which is essential to minimizing the potential for genetic swamp- aster, seed germination, tissue culture, vegetative propagation ing when producing plants for ex situ con- Abstract. Pityopsis ruthii is an endangered species endemic to the Hiwassee and Ocoee servation and restoration. Rivers in Tennessee. As part of a recovery effort focused on P. ruthii, vegetative Previous efforts to restore Ruth’s golden propagation and in vitro multiplication and seed germination techniques were de- aster in a suitable, unoccupied habitat were veloped. Plants were vegetatively propagated using greenhouse stock plants and wild- unsuccessful. Cruzan and Beaty (1998) per- collected stems. Rooting occurred with and without auxin treatments but was greatest formed several experimental plantings, but when 0.1% indole-3-butyric acid (IBA) talc was applied to the vegetative cuttings; after two growing seasons, almost 100% of rooting was lowest when flowering stems were used. Pro-Mix BX substrate provided the the transplants had died. The reasons for the most consistent rooting. In vitro multiplication was accomplished by the removal of experimental failures were not entirely clear, lateral shoots from in vitro-grown plants that were rooted on Murashige and Skoog but the investigators recognized the potential (MS0) basal medium with 270 clones produced from a single individual after 4 months. for drought stress and soil disturbance to Nineteen clones were transplanted and secured with bonded fiber matrix into their negatively impact reintroduced plants. Al- natural habitat and 14 survived for 1 year. To avoid genetic swamping of native though the relative importance of these two populations with the introduction of large numbers of genetically identical individuals factors in the survival of transplanted Ruth’s through clonal propagation, seed-based propagation efforts were explored. Open- golden aster is unknown, some attempt to pollinated seeds were collected, disinfested and germinated, and seedlings established mitigate for drought stress and soil distur- on MS medium. Seeds were submersed in 70% ethanol for 1 minute and briefly flamed. bance will be integral to restoring the species Seeds were surface-sterilized in a range [10% to 50% (v/v)] CloroxÒ bleach solutions with into a suitable habitat. vigorous shaking for 20 minutes, rinsed three times in sterile water, and germinated on The goal of this study was to refine the MS0. Removal of pappus from seeds was required for successful disinfestations, but the standard seed germination protocol, in vitro bleach concentration was not critical. Successful propagation is a step toward the seed germination methodology, and vegeta- conservation and recovery of P. ruthii and should allow future reintroduction projects. tive propagation techniques, including in vitro multiplication of cloned plantlets, to facilitate ex situ conservation and develop- The genus Pityopsis is comprised of seven slopes. The most recent census of Ruth’s ment of a new methodology for restoring perennial species and can be found in the golden aster populations counted 11,150 Ruth’s golden aster into suitable habitat. eastern United States, southeast Mexico, plants; more than 90% occur along the Bahamas, and Central America (Belize, Hiwassee River (Tennessee Valley Author- Materials and Methods Guatemala, and Honduras) (Semple, 2006). ity, unpublished data). Pityopsis ruthii (Ruth’s golden aster) is listed Despite its critically imperiled status, as Plant material. Clones of P. ruthii from under the Endangered Species Act (ESA) and evidenced by its small overall population and an individual plant growing at the University occurs only along two small reaches of the narrow geographical range, there has been of Tennessee were regenerated from leaf and Ocoee and Hiwassee Rivers in Polk County, relatively little applied research focused spe- receptacle tissues using the methods of Wadl TN. It grows in crevices on exposed phyllite cifically on species recovery. However, the et al. (2011) and maintained in vitro and in and graywacke rocks that are in and between U.S. Fish and Wildlife Service, which is the vivo. Open-pollinated seeds (achenes) were the river channel and the adjacent forested federal agency charged with safeguarding collected from plants growing at the University 194 HORTSCIENCE VOL. 49(2) FEBRUARY 2014 PROPAGATION AND TISSUE CULTURE of Tennessee and the Hiwassee and Ocoee Rivers and dried in paper bags at ambient temperature in the laboratory for 3 d. Filled and unfilled seeds were sorted and separated and filled seeds placed into glass vials or seed envelopes, sealed in zip-loc style bags, and stored at 4 °C until use. Filled seeds are differentiated from unfilled seeds as shown in Figure 1. Seed germination. In one experiment, 100 filled seeds, each from four different sites on the Hiwassee River, were obtained from the North Carolina Botanical Garden’s long-term seed storage and germinated in Feb. 2010. Unfortunately, there are no re- cords for baseline germination rates or treat- ment of seeds before long-term storage. Seeds from three of the sites were collected in 1994; seeds from the fourth site were collected in 1995. In the second experiment, seeds were collected from three sites on the Ocoee River and one site on the Hiwassee River in Oct. 2010 and germinated in Nov. 2010. In both experiments, seeds were germi- nated on moist filter paper in petri dishes in the dark between 16 to 24 °C (Farmer, 1977). Daily observations were made and germinated seeds were established in pots filled with Pro- Mix BX and maintained in a greenhouse. In vitro seed germination. Preliminary experiments were conducted to germinate seeds in vitro. To disinfest the seeds, 50 seeds were submersed into 70% ethanol for 1 min and briefly flamed by passing through an Fig. 1. (A–B) Pityopsis ruthii filled and unfilled seeds (achenes). Filled seeds are distinguished from unfilled seeds by the swollen appearance and contained a mature embryo. ethanol burner. This was done to remove as much pappus as possible from the seeds. Seeds were then placed into 50-mL conical 0.8% (w/v) phytagar (Invitrogen, Carlsbad, placed into a 60 · 60-mm petri plate con- centrifuge tubes containing 40 mL of a 20% CA). The pH of the medium was adjusted taining 10 mL of MS0. Plates were incubated (v/v) CloroxÒ bleach shaken vigorously for to 5.8 before autoclaving at 121 °Cand in the dark between 22 and 25 °C for 3 weeks 20 min and then rinsed three times with 103.5 kPa for 20 min. The plates were in- and checked daily for contamination and sterile water. Individual seeds were placed cubated in the dark between 22 and 25 °C. All germination. Germination was considered into 60 · 60-mm petri plates containing seeds were contaminated with unidentified successful after a radicle emerged. 10 mL of MS basal medium (Murashige and fungal and bacterial growth within 48 h of Vegetative propagation. Open-pollinated Skoog, 1962) supplemented with 2% (w/v) culture. seeds that were collected from four locations sucrose, vitamins (2.0 mg·L–1 L-glycine, We suspected that the major source of on the Hiwassee River were obtained from 0.5 mg·L–1 nicotinic acid, 0.5 mg·L–1 pyro- fungal and bacterial contamination in the the North Carolina Botanical Garden and dixine, and 1.0 mg·L–1 thiamine HCl), preliminary experiments originated from the germinated following the methods of Farmer 100 mg·L–1 myo-inositol, and solidified with remaining pappus that was attached to the (1977) and four plants were maintained as seed.