The genus Sarracenia forms a group of carnivorous pitcher plants found mainly in North America. Pitcher plants are found in bog environments throughout the United States, usually in areas of slow-moving water where the acidic soil is poor in nutrients. Pitcher plants use deep tube-like fluid-filled leaves (pitchers) to attract and capture prey helping the plant to compete in their nutrient-poor habitat (Wakefield et al., 2005).
With habitat destruction levels at 93% to 97%, populations have been reduced by 90% and many U.S. pitcher plants have become rare and endangered (Stiefel, 2000; Uhnak, 2003). Three species of this genus, S. leucophylla Raf., S. oreophila (Kearney) Wherry, and S. purpurea spp. venosa (Raf.) Wherry, are considered to be endangered in the state of Georgia and vulnerable in other states (Chafin, 2007; Georgia Department of Natural Resources Wildlife Resources Division, 2011; NatureServe, 2011). Sarracenia oreophila is listed as federally endangered by the U.S. Fish and Wildlife Service (USFWS, 1994). The large interest in carnivorous plants has also had some adverse effects. Carnivorous plants have become popular among hobby horticulturists, fueled in part by soaring Internet sales. Although many species are endangered, plant poachers still harvest wild plants illegally to sell to collectors (Stiefel, 2000). Several Sarracenia species have shown medicinal and commercial value for anticancer treatment (Miles et al., 1974), betulinic acid production for regulation of hair growth (Bradbury et al., 2002), and production of insect attractants (Howse, 1996) leading to added collection pressure on natural populations.
Sarracenia species can be easily grown from seed or vegetatively propagated from rhizomes (Thomas, 2002). However, the process is slow and may yield small numbers of plants. Several books have been devoted to carnivorous plant ecology and cultivation (D'Amato, 1998; Rice, 2006). Reports of seed germination percentages are highly variable ranging from 5% in nature for S. purpurea (Ellison and Parker, 2002) to 85% germination in the light for S. purpurea var terrae-novae de la Pylaie in a greenhouse setting after 32 d of stratification at 4 °C for 6 weeks (Gotsch and Ellison, 1998). In a study that compared taxonomic characters among species, Ellison (2001) examined germination requirements of eight Sarracenia species. Using a 4- to 5-week stratification treatment at 4 °C, germination ranged from 8% to 16% after 34 d for the eight species. Sarracenia leucophylla showed 12% germination and S. purpurea ssp. purpurea and S. purpurea spp. venosa var. burkii showed 10% and 18% germination, respectively.
In vitro propagation from seed, shoot tips, and even rhizomes can multiply the number of individuals of an endangered species rapidly and year round (Fay, 1992, Reed et al., 2011). Once enough plants are available, they can be used to repopulate existing populations or to establish new planting sites for conservation, research, education, or recreational purposes. In vitro-grown plants can be sold to collectors decreasing the collection pressures on wild populations. Although several plant tissue culture companies produce pitcher plants for sale, very little literature is available concerning the propagation of Sarracenia plants using micropropagation techniques. Several researchers have attempted to develop in vitro propagation methods for Sarracenia species (Arnold, 1989; Uhnak, 2003).
Cryopreservation is emerging as a reliable process for seed conservation and long-term storage of many desiccation-tolerant seeds and tissues (Englemann, 2011; Pence, 2011; Pritchard, 2007; Reed et al., 2011). Genetic preservation through long-term seed storage is indicated as a goal in the recovery plan for endangered Sarracenia oreophila (USFWS, 1994). Seed cryopreservation may play an important role in the conservation of these rare and endangered plants.
The objectives of this study were to develop a reliable micropropagation protocol for endangered Sarracenia species using seed to start cultures. A further objective was to investigate the effect of seed cryostorage on these endangered species.
Adams, R.M. II, Koenigsberg, S.S. & Langhans, R.W. 1979 In vitro propagation of Cephalotus follicularis (Australian pitcher plan) HortScience 14 512 513
Ahmed, E.U., Hayashi, T. & Yazawa, S. 2004 Auxins increase the occurrence of leaf-color variants in Caladium regenerated from leaf explants Sci. Hort. 100 153 159
Arnold, A. 1989 Propagation of Sarracenia oreophila (Kearney) Wherry by seeds, leaf cuttings, and tissue culture MS thesis, University of Georgia Atlanta, GA
Bradbury, B.J., Soper, S.J., Kaczvinsky, J.R., Bailey, D.L. & Gale, C.D. 2002 Compositions which contain triterpenes for regulating hair growth US Patent No. 6,482,857.
Choi, S.Y. & Han, N.Y. 2004 In vitro mass propagation medium of Sarracenia species and method for mass production thereof using the same Korean Patent No. 204102548.
Ellison, A.M. 2001 Interspecific and intraspecific variation in seed size and germination requirements of Sarracenia (Sarraceniaceae) Amer. J. Bot. 88 429 437
Ellison, A.M. & Parker, J.N. 2002 Seed dispersal and seedling establishment of Sarracenia purpurea (Sarraceniaceae) Amer. J. Bot. 89 1024 1026
Georgia Department of Natural Resources Wildlife Resources Division 2011 <http://www.gohuntgeorgia.com/content/protectedplants.asp>.
Gotsch, S.G. & Ellison, A.M. 1998 Seed germination of the northern pitcher plant, Sarracenia purpurea Northeastern Naturalist 5 175 182
Latha, P.G. & Seeni, S. 1994 Multiplication of the endangered Indian pitcher plant (Nepenthes khasiana) through enhanced axillary branching in vitro Plant Cell Tissue Organ Cult. 38 69 71
MacKay, J., Becwar, M., Park, Y., Perfetti, C., Cordero, J., Lockart, L. & Pullman, G.S. 2006 Genetic control of somatic embryogenesis initiation in loblolly pine and implications for breeding Tree Genet. Genomes 2 1 9
Miles, D.H., Kokpol, U., Zalkow, L.H., Steindel, S.J. & Nabors, J.B. 1974 Tumor inhibitors. 1. Antitumor activity of Sarracenia flava J. Pharm. Sci. 63 613 615
Murashige, T. & Skoog, F. 1962 A revised medium for rapid growth and bioassays with tobacco tissue cultures Physiol. Plant. 15 473 497
Pence, V.C. 2011 Evaluating costs for the in vitro propagation and preservation of endangered plants In Vitro Cell. Dev. Biol. Plant 47 1 4
Pritchard, H.W. 2007 Cryopreservation of desiccation-tolerant seeds 185 201 Day J.G. & Stacey G.N. Methods in molecular biology, Vol. 368: Cryopreservation and freeze-drying protocols 2nd Ed Humana Press Inc. Totowa, NJ
Pullman, G.S., Chopra, R. & Chase, K.-M 2006 Loblolly pine (Pinus taeda L.) somatic embryogenesis: Improvements in embryogenic tissue initiation by supplementation of medium with organic acids, Vitamins B12, and E Plant Sci. 170 648 658
Reed, B.M., Sarasan, V., Kane, M., Bunn, E. & Pence, V.C. 2011 Biodiversity conservation and conservation biotechnology tools In Vitro Cell. Dev. Biol. Plant 47 1 4
Stiefel, C.F. 2000 Meat-eating plants Science World. 20 Mar. 2000. <http://findarticles.com/p/articles/mi_m1590/is_12_56/ai_61291780/>.
Uhnak, K.S. 2003 Micropropagation of carnivorous plants MS thesis, ETD Collection for University of Rhode Island. Paper AAI3103728.
Van Winkle, S. & Pullman, G.S. 2003 The combined impact of pH and activated carbon on the elemental composition of plant tissue culture media Plant Cell Rep. 22 303 311
Wakefield, A.E., Gotelli, N.J., Wittman, S.E. & Ellison, A.M. 2005 Prey addition alters nutrient stoichiometry of the carnivorous plant Sarracenia purpurea Ecology 86 1737 1743
Walls, M. 2009 Seed storage guidelines for California native plant species <http://www.rsabg.org/documents/horticulture/Seed%20Collecting%20and%20Storage%20Guidelines.pdf>.
Xu, G., Chen, J., Wang, Y., Lv, Y. & Mou, H. 2009 Aseptic seeding and tissue culture method for rapid propagation of Sarracenia Faming Zhuanli Shenqing Gongkai Shuomingshu. 5 pp.