CryoLetters 29(4), 271-276 (2008) CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK SEED CRYOPRESERVATION OF Halimium AND Helianthemum SPECIES Félix Pérez-García* and M. Elena González-Benito Departamento de Biología Vegetal, Escuela Universitaria de Ingeniería Técnica Agrícola, Universidad Politécnica de Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain E-mail: [email protected] Abstract Seed germination of four Halimium species [H. atriplicifolium (Lam.) Spach, H. halimifolium (L.) Willk., H. ocymoides (Lam.) Willk., H. umbellatum (L.) Spach ssp. viscosum (Willk.) O. Bòlos & Viso] and eight Helianthemum species [H. almeriense Pau, H. appeninum (L.) Mill., H. cinereum (Cav.) Pers., H. hirtum (L.) Mill., H. marifolium (L.) Mill., H. nummularium (L.) Mill., H. syriacum (Jacq.) Dum. Cours., H. squamatum (L.) Dum. Cours.], all Cistaceae species from the Mediterranean region, was studied after seed storage in liquid nitrogen (LN, -196ºC) for four months. In all samples assayed, mechanical scarification of the seed coat was carried out to enhance seed germination. For most samples studied, final germination percentages were unaffected by storage of seeds in LN, both for intact seeds and for scarified seeds. The germination rate of cryopreserved seeds, expressed as days to reach 50% of the final germination percentage (T50), was lower only for four samples of intact seeds and for three samples of scarified seeds. Therefore, this study shows that seed cryopreservation could be a suitable procedure for the long-term seed conservation of several Halimium and Helianthemum species. Keywords: Cistaceae, cryopreservation, liquid nitrogen, seed germination, seed scarification INTRODUCTION Five genera of the family Cistaceae (Cistus, Fumana, Halimium, Helianthemum and Xolantha) form an important part of the Mediterranean shrub (21). In the present work, four Halimium species [H. atriplicifolium, H. halimifolium, H. ocymoides (2 populations) and H. umbellatum (2 populations)] and eight Helianthemum species [H. almeriense, H. appeninum (3 populations), H. cinereum, H. hirtum, H. marifolium, H. nummularium, H. syriacum and H. squamatum] were tested for seed cryoconservation. Three of the twelve species studied (Halimium atriplicifolium, Helianthemum almeriense and Helianthemum squamatum) are Iberian endemic shrubs. All shrubby species studied could be used as ornamental plants and they show a remarkable plasticity and adaptability to rather extreme ecological conditions (cold, aridity, poor soils) and therefore they are of interest because of their potential use to restore arid zones. 271 Seed storage is the simplest and most efficient method for ex situ conservation of plant genetic resources. Conventional seed banks have traditionally been used for germplasm conservation. There are reports on successful long term seed conservation of wild species in seed banks (3, 10, 18). However, in those storage conditions some seed deterioration may occur (20). Seed cryopreservation has emerged as a option for ex situ long-term conservation of wild species, especially for small, orthodox seeds (1, 2, 4, 6, 7, 8, 9, 13, 15, 23). González- Benito et al. (7) proved that seed cryopreservation is useful for germplasm conservation of three Cistaceae species (Cistus osbeckiifolius, Helianthemum polygonoides and Helianthemum squamatum). An additional problem in seed preservation of wild species is that many of them show seed dormancy. Thus, in such cases, treatments are needed to allow germination in laboratory conditions. Seed coat hardness and impermeability to water are the most important causes of the low germination of several Halimium and Helianthemum species (5, 12, 14, 16, 17, 19, 21, 22). However, seed germination can be enhanced, in the laboratory, by mechanical scarification (5, 16, 17, 19). Therefore, in all species studied, mechanical scarification was also carried out and their possible interaction with cryopreservation was studied. The aim of this research was to evaluate the effect of seed cryopreservation on the germination of several Halimium and Helianthemum species. MATERIALS AND METHODS For each accession, ripe fruits (capsules) containing mature seeds were collected in the field in July 2002 and July 2003. Table 1 shows the collection year and collection site, as well as the ecological preference of the 16 accessions collected. The seeds from all collected capsules showed a similar degree of ripeness, as it was observed from their colour and hardness. For each species, fruits were randomly chosen from 20 different plants before extracting the seeds from the capsules. Seeds were cleaned, kept in hermetic glass containers and stored in a dry place (ca. 30% RH) at room temperature (ca. 23ºC) until the start of the trials in the autumn of the same year that the seeds were collected. The seed water content, expressed as a percentage of fresh weight, was determined using the air oven method (103ºC for 17 h; 11). The seed water content determination was carried out immediately prior to seed storage. Seed samples were placed in 2 ml plastic cryovials and immersed in liquid nitrogen (LN, -196ºC, seed cooling rate of 130ºC min-1; 9) for four months. The subsequent warming procedure consisted of removing the samples from cryogenic storage and allowing them to warm under room temperature (ca. 23ºC) to equilibrium (24 h). The seeds were then tested for germination. For cryopreserved seeds, mechanical scarification was performed after thawing. Thus, in all samples assayed, mechanical scarification of the seed-coat was carried out in one group of cryopreserved seeds in order to enhance seed germination. Seeds were mechanically scarified by abrasion between two sheets of fine-grained sandpaper. In all trials, four replicates of 25 seeds each were tested for germination on top of two sheets of filter paper (previously moistened with 3.5 ml distilled water) in 7-cm diameter glass Petri dishes. The Petri dishes were randomly placed in a seed germinator at 15ºC under a 16 h light / 8 h dark photoperiod. The incubation temperature chosen (15ºC) was based on previous experiences (5, 19). Distilled water was added as needed to maintain constant moisture during the experiments, and germinating seeds were counted and removed every two days over a 60- day incubation period. The criterion for germination was emergence of the radicle through the seed coat. Seed samples used as controls followed the same procedure described for cryopreserved seed samples, except for the LN storage itself. 272 Table 1. Seed collection site, collection year and ecological preference of Halimium and Helianthemum samples assayed. Species Collection site (Province) and Distribution and ecological collection year preference Halimium atriplicifolium Arganda (Madrid, central Spain). Endemic shrub of central 2002 and S of Spain Halimium halimifolium El Saler (Valencia, E Spain). 2002 W Mediterranean shrub preferably on sandy soils Halimium ocymoides population 1 Badajoz (Badajoz, SW Spain). 2003 Shrub on siliceous soils of population 2 Guadalupe (Cáceres, SW Spain). W and central Iberian 2003 peninsula and N of Africa Halimium umbellatum population 1 Robledillo de Trujillo (Cáceres, SW Central and S Iberian Spain). 2003 Peninsula and N of Africa, population 2 Guadalupe (Cáceres, SW Spain). preferably on acid soils 2003 Helianthemum Sorbas (Almería, SE Spain). Endemic gypsophyte shrub almeriense 2002 of arid SE Spain Helianthemum appeninum population 1 El Arenal (Ávila, central Spain). 2002 Mediterranean shrubby population 2 Somiedo (Asturias, NW Spain). 2002 vegetation on rocky soils population 3 Entrepeñas (Guadalajara, central Spain). 2003 Helianthemum Albacete (Albacete, SE Spain) Central and W cinereum 2003 Mediterranean basin, on calcareous soils Helianthemum hirtum Pinilla del Valle (Madrid, central, SW Europe and N Africa, Spain). 2002 preferably on calcareous or gypsum soils Helianthemum El Saler (Valencia, E Spain). W Mediterranean shrub on marifolium 2002 calcareous or gypsum soils Helianthemum Somiedo (Asturias, NW Spain). Shrub on calcareous or nummularium 2002 siliceous soils of Europe and W Asia Helianthemum Rivas (Madrid, central Spain). 2003 Iberian endemic shrub squamatum widely distributed on gypsum soils Helianthemum El Saler (Valencia, E Spain). 2002 Mediterranean shrubby syriacum vegetation on calcareous or gypsum soils and sandy soils At the end of the incubation period (60 days), the final germination percentage and the time required (in days) to reach 50% of the final germination percentage (T50) were scored. When the final germination percentage was equal to or less than 5%, the T50 value was not calculated. The germination percentage values were arcsine transformed and then subjected to one-way analysis of variance. The statistical analysis of T50 values was also carried out using one-way ANOVA. Results of the mechanical scarification treatments were analysed separately. 273 RESULTS Seed water content ranged from 3.9% to 4.7%. Table 2 shows final germination percentages after cryopreservation period of four months for all species studied. In all samples assayed, mechanical scarification of seed coat significantly increased germination in both control (non-cryopreserved seeds) and cryopreserved seeds (Table 2). Table 2. Final germination percentages for Halimium and Helianthemum intact and scarified seeds after storage in liquid nitrogen (LN) for four months. Control = non-cryopreserved seeds. P: for each sample, significance of control vs. cryopreserved