HORTSCIENCE 48(6):750–755. 2013. extirpated. Extant populations typically have 10 to 500 stems often containing only one or a few genotypes (Federal Register, 2009; Seed Cryostorage and Micropropagation USFWS, 2010). Georgia aster grows up to 100 cm in of Georgia Aster, Symphyotrichum height and flowers in early October to mid- November with 5-cm diameter heads having georgianum (Alexander) Nesom: dark purple rays surrounding white (occa- sionally purple) disc flowers with purplish tips, purplish anthers, and whitish pollen A Threatened Species from the (Chafin, 2007; Georgia Department of Natu- ral Resources Wildlife Resources Division, Southeastern United States 2008; Weakley, 2011). Successful pollina- tion results in the production of achene-like Sullivan Lynch and Rachel K. Johnston fruits (cypselae) that each contain a single School of Biology, Georgia Institute of Technology, 310 Ferst Drive, Atlanta, seed and embryo. However, the species re- GA 30332 produces mostly asexually from rhizomes (USFWS, 2010). The habitat it now most Ron O. Determann and Jennifer M. Cruse-Sanders often occupies include roadsides, utility rights- Department of Conservation Research, Atlanta Botanical Garden, 1345 of-way, and openings where land manage- Piedmont Avenue, NE, Atlanta, GA 30309 ment mimics the roles formerly played by fire and a dry upland habitat. Unfortu- Gerald S. Pullman1 nately, these areas also leave Georgia aster School of Biology, Georgia Institute of Technology, Atlanta, GA 30332; and the at risk to invasive species such as kudzu, Institute of Paper Science and Technology, Georgia Institute of Technology, herbicides, road expansion, and develop- ment. Herbicide use can further fragment 500 10th Street NW, Atlanta, GA 30332-0620 populations, potentially reducing popula- Additional index words. conservation, cryopreservation, endangered species, Georgia aster, tion persistence and survival in small iso- micropropagation, Symphyotrichum georgianum lated patches. In vitro propagation from seed, shoot tips, Abstract. Symphyotrichum georgianum (Asteraceae), commonly known as Georgia aster, and rhizomes can multiply the number of is a candidate for listing under the Federal Endangered Species Act in the four individuals of an endangered species rapidly southeastern U.S. states where it lives. Rarity of this species is thought to be attributable and continually (Fay, 1992; Reed et al., 2011). in part to small population sizes and limited seed production. Protocols for in vitro Once enough plants are available, they can germination, sustainable shoot micropropagation, shoot establishment in soil, and seed be used to repopulate old populations or to cryopreservation are presented that will assist in the safeguarding and augmentation of establish new populations for conservation, dwindling natural populations. Germination in vitro on growth regulator-free half-strength research, education, or recreational purposes. Murashige and Skoog (MS) medium after sterilization in H2O2 initiated the development of Cryopreservation is emerging as a reliable shoot cultures. Shoot multiplication and elongation occurred on half-strength MS salts process for seed conservation and long-term containing 0.1 mg·L–l benzylaminopurine and 0.2 mg·L–l gibberellic acid, producing an storage of many desiccation-tolerant seeds average of 18 new shoots over a 6- to 8-week subculture cycle. Shoots rooted easily when and is likely to maintain seed viability for planted into cutting mix after treatment with rooting powder containing indole-3-butyric longer periods than conventional storage at acid (IBA) or in vitro rooting in medium with or without N-acetyl-L-aspartic acid (NAA). –18 °C (Englemann, 2011; Li and Pritchard, Plant survival after 1 month was 90% or higher for all treatments. Cryopreservation tests 2009; Pritchard, 2007; Reed et al., 2011). Seed with seeds from three populations averaged 46.7% germination compared with control seed cryopreservation may play a particularly im- (no cryostorage) germination of 43%; differences were not statistically significant. Fresh portant role in the conservation of threatened seeds and seeds equilibrated for 1 to 4 weeks at room temperature and 12% relative plants because safeguarding or propagating humidity did not differ significantly in germination post-cryopreservation. Initial observa- material from seed maintains new genetic tions suggest that Georgia aster rapidly loses seed viability over 1 to 2 years when stored at combinations resulting from sexual reproduc- room temperature. The ability to increase seed longevity through cryopreservation storage tion (Pence, 1991). may be a critical step in the conservation of this species. The objectives of this study were to develop a reliable micropropagation proto- col for threatened Georgia aster using seeds Symphyotrichum georgianum (Alexander) of the sunflower family (Asteraceae). Georgia to start cultures and to investigate the feasi- Nesom (syn. Aster georgianus), commonly aster is native to open, grassy meadow habitats bility of long-term seed storage through known as Georgia aster, is a perennial member and has been steadily losing its natural habitat cryopreservation. mainly as a result of forest fire prevention and extirpation of large native grazing animals. It Received for publication 20 Mar. 2013. Accepted is now classified as a vulnerable species with Materials and Methods for publication 3 Apr. 2013. a moderate and imminent threat of extinction We thank Allyson Reed, Joanne Baggs, and Robyn (Federal Register, 2009; USFWS, 2010). The Plant materials, experimental design, Mackie for help in obtaining Georgia aster seed and species is currently found in south–central and evaluation gratefully acknowledge the help of Kylie Bucalo, North Carolina, South Carolina to central A small number of S. georgianum seeds Maria Dinh, Dionne Wells, and Kathryn Wright. Georgia, west to central Alabama, and on the collected in Nov. 2009 from two locations We thank the Georgia Institute of Technology for Coastal Plain of southwest Georgia. There designated as CP and PM in Cobb County, providing a Materials, Supplies and Travel Grant for are 127 known populations, many of which GA, were stored at 22 to 25 °C until germi- Undergraduate Research in support of this project and the USDA Forest Service for partial funding of have not been seen in 10 years and 23 of nation tests occurred in 2010. Seeds were also the study. Seed was obtained under National Park which have been either extirpated or not collected in Dec. 2010 from three Union Service permit # KIMO-2011-SCI-003. observed for over 20 years (USFWS, 2010; County, SC, sites (SC5, SC7, SC8), stored 1To whom reprint requests should be addressed; Weakley, 2011). Historic populations in the at 22 to 25 °C, and used during winter and e-mail [email protected]. eastern Panhandle of Florida are considered Spring 2011. 750 HORTSCIENCE VOL. 48(6) JUNE 2013 Georgia aster seed sterilization and washed with a solution of 10% Liqui-Nox and plates and kept at 25 to 26 °C under a 16/8-h germination in vitro and after storage 0.2% Tween 20 for 10 min, rinsed in running (day/night) photoperiod with light from cool Preliminary experiments with small num- cold tap water for 30 min, and then surface- white fluorescent lamps at 30 mmol·m–2·s–1. bers of 2009 CP and PM seeds indicated that sterilized aseptically in 20% H2O2 for 10 or 15 Germination responses were evaluated at 3 germination occurred on one-third strength min with continuous stirring. Seeds were then weeks. MS salts (Murashige and Skoog, 1962), the rinsed five times with sterile deionized water Storage. To test seed viability after long- large seed pappus did not need to be removed (Pullman et al., 2005, 2006). Seed numbers term storage, 38 filled seeds from CP col- during seed sterilization, filled seed should be used from the two collections (CP and PM) lected in Nov. 2009 and stored in a plastic bag selected using a strong light behind the seeds, were 18 and 10, respectively, for each treat- at 4 °C for 2 years and 30 filled seeds from and stronger sterilization than 10 min in 20% ment. Seeds were placed individually onto SC8 collected in Dec. 2010 and stored in H2O2 (Pullman et al., 2005, 2006) was needed. 7 mL of semisolid one-third-strength MS a plastic bag at 22 to 25 °C for 1 year were Germination and sterilization. Year 2009 salts along with 3% sucrose, 100 mg·L–1 tested for germination after surface steriliza- –1 seeds were examined with a strong light, and myo-inositol, 0.33 mg·L thiamine HCl, tion in 20% H2O2 for 15 min and 3 weeks on empty seeds were discarded (Fig. 1A). To 0.165 mg·L–1 pyridoxine HCl, 0.165 mg·L–1 growth regulator-free half-strength MS salts reduce seed surface fungal and bacteria con- nicotinic acid, 0.66 mg·L–1 glycine, and with 3% sucrose, 100 mg·L–1 myo-inositol, tamination seen in preliminary experiments, 4.5 g·L–1 Phytagel. Medium pH was ad- 0.495 mg·L–1 thiamine HCl, 0.248 mg·L–1 full seeds with attached pappus were split into justed with KOH or HCl to 5.7 after addition pyridoxine HCl, 0.248 mg·L–1 nicotinic acid, –1 –1 two treatments, and sterilization in 20% H2O2 of all ingredients except gelling agent and 0.99 mg·L glycine, and 4.5 g·L Phytagel for 10 or 15 min was compared. Seeds were autoclaved at 121 °C for 20 min. Seeds were with pH adjusted to 5.7. Seeds from the same washed in running cold tap water for 10 min, incubatedonmediumin603 15-mm petri lots, 23 from CP and 58 from SC8, were tested for germination in vitro shortly after collection using the same methods. Georgia aster growth in vitro and shoot multiplication experiments Nutritional salts. To determine the opti- mal strength of MS salts, shoot growth was compared across one-third strength, half- strength, and full-strength MS medium with- out growth regulators.