CryoLetters 26 (2), 121-130 (2005) © CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK SURVIVAL OF FOUR ACCESSIONS OF Anigozanthos manglesii (HAEMODORACEAE) SEEDS FOLLOWING EXPOSURE TO LIQUID NITROGEN D. J. Merritt1*, D.H. Touchell2, T. Senaratna1,3, K.W. Dixon1,3 and C. W. Walters4 1Kings Park and Botanic Garden, West Perth, WA 6005, Australia. 2School of Forestry and Wood Products, Michigan Technological University, Houghton, MI 49931-1295, USA. 3Faculty of Natural and Agricultural Sciences, The University of Western Australia, Crawley, WA, 6009, Australia. 4USDA-ARS National Centre for Genetic Resources Preservation, 1111 South Mason St, Fort Collins, CO 80521, USA. *Correspondence: [email protected] Abstract This study investigated the survival of seeds from the prominent endemic Western Australian species Anigozanthos manglesii following exposure to liquid nitrogen (cryostorage). Seeds from four different accessions (collected in 1987, 1990, 1993 and 1998) adjusted to different water contents were tested for survival following cryostorage. Water content was a significant determining factor with survival of cryostored seeds declining rapidly at water contents above c. 18%. These water contents were deemed as critical water contents and were supported by DSC scans showing high endothermic peaks indicating ice crystallisation. In some instances, survival of cryostored seeds also declined at low water contents. Seeds from 1990 had a lower than expected survival compared to the other accessions. This may have resulted from the higher lipid content of seeds from this accession, or the reduced germination and vigour of these seeds prior to cryostorage. Keywords: Anigozanthos manglesii, conservation, cryostorage, seed, water content. INTRODUCTION Seed banks for conservation purposes must ensure the long-term survival of an accession. Seeds of wild species may be in short supply and, for threatened species in particular, there may be limited collection opportunities and few seeds available to form a collection. Therefore, for valuable germplasm it is essential to provide the best possible storage conditions as any loss of seed viability during storage may impact significantly on the number of plants available for restoration projects in future years. Cryostorage offers an ideal method for germplasm conservation as there is the potential for extended storage periods (possibly many hundreds or thousand of years) with no loss of viability (13,27). The development of methods for cryostorage of seeds initially centred on species of agricultural importance (10,12,20). More recent studies have demonstrated the potential of cryostorage for conservation of wild species (4,7-9,17,18) and cryostorage is viewed as an important tool for the conservation of priority species in Australia (19). Seed survival following cryostorage is highly dependent upon seed water content. Many studies have documented the existence of a critical water content (sometimes termed the high moisture freezing limit) below which successful cryostorage is not constrained by cooling rates or addition of cryoprotectants. Exposing seeds to liquid nitrogen above the critical water content results in lethal ice crystal formation (2,10,14,21). Some species also show damage at water contents much lower than the critical threshold associated with ice-crystal formation 121 (7,13,20,27). Therefore, resolving the optimal water content(s) is essential for successful cryostorage. The effects of seed age on survival of cryostorage have received less attention, although a previous study on Banksia ashbyi demonstrated that freshly collected seeds were more able to survive cryostorage at water contents exceeding the critical threshold, compared to older seeds (7). Many seed banks have seed accessions that have been stored under conventional conditions for a number of years. For greater security and longevity of important germplasm (e.g. that of rare and endangered species) it may be beneficial to transfer these collections to cryostorage to maximise longevity, provided the seed quality is not adversely affected by such treatment. This study investigates cryostorage of seeds of Anigozanthos manglesii D. Don (Haemodoraceae), a species representative of an important endemic Western Australian genus comprising 25 species, nine of which are currently stored in the seed bank at Kings Park and Botanic Garden, Western Australia, with accessions dating back to 1972. Specifically, this study examines: the ability of seeds from different accessions to survive cryostorage; whether the critical water content, above which survival is significantly reduced, varies between seeds from different accessions; whether variations in survivability and the critical water content are related to seed lipid content; and, for seeds of one accession, thermal transitions and electrolyte leakage in relation to survival following cryostorage at different water contents. MATERIALS AND METHODS Seed material Seeds of Anigozanthos manglesii from four different accessions were obtained from the seed store at Kings Park and Botanic Garden, in Perth, Western Australia. Seeds collected in 1987, 1990, 1993 and 1998 were selected. Prior to the experiments in this study (conducted in 1998 and 1999), seeds collected in 1987 and 1990 were stored air-dry at c. 23°C and seeds collected in 1993 were stored at -14°C. Seeds collected in 1998 were used immediately. All seeds were collected from within bushland at Kings Park and Botanic Garden. Seed germination testing Freshly collected Anigozanthos manglesii seeds have an after-ripening requirement for germination to proceed. This can be circumvented by heat-pulsing seeds at 100°C for 3 h (16). Prior to any experimentation, all seeds were heat-pulsed to remove the confounding factor of the influence of seed dormancy on germination of seeds of different ages. Following the heat-pulse, seed water content was adjusted by placing seeds over saturated solutions of ZnCl2 (5% RH), LiCl (13% RH), MgCl2 (32% RH), K2CO3 (43% RH), Ca(NO3)2 (50% RH), NaCl (75% RH), KCl (85% RH) and KNO3 (92% RH) (24, 29) at 23°C for 4 weeks. For the highest water contents tested (> 20%) seeds were placed over de-ionised water for varying -1 periods between 7 - 14 h. Seed water content was determined gravimetrically (g H2O g DW) by drying three replicates of 50 seeds in an oven at 103°C for 17 h (6). Following adjustment of seed water content, seeds were removed for immediate germination testing (non- cryostored), or placed in 2 ml polyethylene cryovials (Nunc®), plunged directly into liquid nitrogen and stored for 1 week, then warmed at room temperature for 20 min before further handling. To test for germination, seeds were sterilised in a 2% calcium hypochlorite solution (Ca(OCl)2) for 15 mins, washed in sterile de-ionised water, and incubated on sterile water agar plates (0.7% w/v) in the dark at 18°C. Five replicates of 10 seeds were incubated for each treatment. Seeds were considered germinated upon radicle emergence and germination was scored every 3 d, from the time the first seeds germinated, for 3 weeks, at which time maximum germination had occurred. Total seedling length was measured after 3 weeks. Time to initial germination (t0) and time to 50% maximum germination (t50) were determined from cumulative germination curves derived from the mean values of the five replicates. 122 Seed lipid content Seed lipid content was determined following the methods of Senaratna et al. (11). Three replicates of 50 seeds (at air dry water contents) were homogenised in chloroform:methanol (2:1 by volume) and centrifuged at 12 000 rpm for 15 min. The supernatant was partitioned with 0.7% (w/v) NaCl and centrifuged again at 10 000 rpm for 10 min. The organic layer was -1 evaporated under N2 and residual lipids determined gravimetrically (g lipid g FW). Differential scanning calorimetry For seeds collected in 1993, warming thermograms of seeds at different water contents were measured via DSC analysis, using a Perkin Elmer DSC7. Between 5 - 8 seeds (giving a fresh weight of c. 30 mg) were used for each test. Following adjustment of seed water content, seeds were sealed in aluminium pans and cooled at 10°C min-1 from 20°C to -150°C, then warmed at the same rate (25). Thermal transitions were measured as endothermic (melting) peaks in the baseline and the enthalpy of the transitions calculated as the area under the peak. The onset temperature of a transition was determined by the intersection of the baseline and the tangent to the steepest part of the peak. All calculations were made using Perkin Elmer software. Electrolyte leakage Electrolyte leakage was also measured for seeds collected in 1993. Seeds were heat- pulsed and equilibrated to a range of water contents as described above. Seeds were then cryostored or non-cryostored as for the germination trials and placed in vials containing 20 ml de-ionised water. The conductivity of this solution was measured over a 3 h period using a TPS 900-C conductivity meter. Three replicates of 30 seeds were used for each test. Statistical analysis Germination, seedling length and electrolyte leakage data were analysed by analysis of variance. Germination percentage values were arcsine-transformed prior to statistical analyses (untransformed data appears in all figures). Fisher’s least significant difference (P<0.05) was used to determine significant differences between treatments. RESULTS Seed germination Following adjustment at different relative humidities, seed water content ranged from c. 2 - 26% (Fig.1, Table 1). Germination of non-cryostored seeds collected in 1987, 1993 and 1998 averaged c. 90% (across all water contents). Non-cryostored seeds collected in 1990 had lower germination, averaging 74% (Fig.1a-d). Seed water content did not greatly affect germination of non-cryostored seeds and germination generally varied by ≤10% across all water contents, although in some instances, particularly for seeds collected in 1987 and 1990, a significant increase (P<0.05) in germination was noted at high water contents (17 – 21%), compared to lower water contents.