Seed storage of Himantoglossum robertianum (Orchidaceae): a comparison of temperature and moisture conditions

3.1 = SEED STORAGE OF HIMANTOGLOSSUM ROBERTIANUM (ORCHIDACEAE): A COMPARISON OF TEMPERATURE AND MOISTURE CONDITIONS ANDREA PIRONDINI1, TIMOTHY R. MARKS2, ELISABETTA SGARBI1 1) Department of Life Sciences, University of Modena and Reggio Emilia, Via Amendola 2, Padiglione Besta, 42122 Reggio Emilia, Italy; 2) Seed Conservation Department, Millennium Seed Bank - Royal Botanic Gardens Kew, Wakehurst Place, UK. Seed banks maintained at -20 °C and low relative humidity are routinely used for the long-term ex situ conservation of orthodox seeds (1). Seed longevity, as measured by survival time during storage, is a critical aspect in the management of a seed bank because it may differ significantly between species and accessions (2). Moreover, the effectiveness of seed survival strongly depends on the correct application of standard procedures before seed storage (3). Temperature and seed moisture content are recognized as the main factors that influence the duration over which seeds can be successfully conserved (3). Orchid seeds are considered to display an orthodox behaviour as they show an extended longevity when subjected to a drying treatment (< 5% moisture content) and freezing at -20 ºC, conditions commonly applied in many seed banks (4). It is also known that orchid seeds tolerate different storage conditions, but it is unclear which is the best approach for long-term storage, as differences in viability occur among different orchid species during the storage (5). The aim of this study was to establish the optimal conditions for the conservation of seeds of Himantoglossum robertianum H. Baumann, an Euri-Mediterranean terrestrial orchid (Fig. 1). Investigations were addressed to compare seed storage conditions, 5 °C, -20 °C and -196 °C (storage in liquid nitrogen), after the seed had been equilibrated to a range of relative humidity (RH) conditions, i.e. 5%, 60% and 80% (Fig. 2; 3; 4), constructed using different concentrations of the lithium chloride solutions. Isotherms were determined to describe the relationship between seeds moisture content and RH and relate to data acquired from Differential Scanning Calorimetry. An in vitro germination test was carried out to verify the effectiveness of the different pre-sowing treatments and storage conditions to maintain the seed germination capability. The highest percentage of germination was obtained with the lowest RH in the cryopreserved seeds and at 5 °C storage temperature. Subsequently, we considered the growth of protocorms in order to determine if significant differences in protocorm development occurred within the following five months. The development of H. robertianum protocorms did not start until the third month after sowing, and showed a linear increase among all the treatments. In particular, the cryogenic treatment gave good results in combination with low RH values. 1) S. H. Linington, H. W. Pritchard (2001) in Encyclopedia of Biodiversity, vol 3. Academic Press 2) T. D. Hong , R. H. Ellis (1996) IPGRI, Technical Bulletin No. 1, Rome 3) A. L. Batty, K. W. Dixon, M. Brundrett, K. Sivasithamparam (2001) Austr. J. Bot., 49, 619-628 4) P. T. Seaton, J. P. Kendon, H. W. Pritchard, D. M. Puspitaningtyas, T. R. Marks (2013) Lankesteriana, 13, 93-101 5) P. T., Seaton, H. W. Pritchard (2008) Orchids, 76, 762-773