Banco de Germoplasma de la Universidad Rey Juan Carlos

Study Extent

The Sierra de Guadarrama and gypsophylic steppes of Central Spain are the most significant areas represented in URJC Germplasm Bank. Seeds have been collected from 8 provinces of Spain: Ávila, Cuenca, Guadalajara, Madrid, Salamanca, Segovia, Valencia y Zaragoza.

Sampling

In the collection phase, seed samples were taken from several different localities to cover the distribution range of the communities in the study area and thereby obtain genetic material from the different species with the potential to adapt to different local conditions.
To plan seed collection, it was essential to determine the timing of seed dispersal. In this sense, the literature related to the phenology of the collected species was consulted. Other morphological indicators of the timing of natural dispersal were also used. These included tissue hardness, changes in color in seeds and fruits, dryness of pods and capsules, among others. The physical quality of the seeds was evaluated at the time of collection to avoid collecting specimens that were infected or clearly unviable. We aimed to collect between 3000 and 5000 seeds per accession to guarantee that there is enough material for germination assays, long-term conservation and, if necessary, propagation (Gold et al. 2004). In order not to compromise the viability of the population in situ, no more than 20% of the seeds present at the time of collection were sampled (Gold et al. 2004). Once samples were collected for each species, they were placed in paper bags and labeled with the name of the species, the site where they were collected, altitude, site coordinates, the date of collection and the name of the collector. This information constituted the passport data for each seed accession.

Quality Control

The URJC Germplasm Bank has a protocol which describes the seed processing methods for collection, processing in the laboratory, germination assays, dessication, recording accessions, labeling, scanning and freezing. Seed collectors are professors and researchers with great knowledge of the flora characteristic of each ecosystem. They are all members of research groups that have worked in this field for years and, therefore, have great experience in the phenology and identification of the species. Moreover, some accessions have herbarium specimens. 63% of the records are georreferenced with GPS and radio points. When this information is not available, the geographic coordinates of the site and the extension of the municipality are used as a surrogate of the measure.

Method steps

1. Seed processing in the laboratory: The first treatment consisted of cleaning the seeds. Sieves with stainless steel meshes of different sizes (mesh holes between 2 mm-0.5 cm) were used to separate as much undesired material as possible (soil, stones, small leaves, stems, flowers, etc.) from the seed sample. The separated seeds were then introduced in tubes identified with the passport data (species name, date, site, altitude, collector) and stored in a cupboard at room temperature under ambient moisture conditions. Germination assays: Germination assays were carried out with 100 seeds of each accession. Four replicates of twenty-five seeds were placed in Petri dishes on two pieces of filter paper. Distilled water was added until the surface was wet and the dishes were placed in a germination chamber (Selecta Hotcold GL, Barcelona, Spain). Accessions from the high mountain collection were incubated at 15ºC with a 16/8 hour light/dark photoperiod (Giménez-Benavides et al. 2005), while accessions from the semi-arid collection were incubated at 20ºC with the same photoperiod (Pérez-García and Durán 1989, Escudero et al. 1997, Herranz et al. 2002). Assays were monitored every 2-3 days for a minimum period of one month, and the assay was considered to have finished when no germination was observed for four subsequent censuses (8-day period). Germinated seeds were counted and removed in each census. Dessication: Silica gel (SiO2) was used to desiccate the samples. As the desiccation rate increases with the amount of gel used, the base of the desiccator was filled to a 1:1 proportion with the seeds. The seeds which were separated by accessions in the Petri dishes were placed uncovered on the metal plate in the desiccator. They were then placed in a cool place to dry for at least two months. Recording accessions: Accessions were added to the database of the URJC Germplasm Bank using Herbar Zoorbar Ligero (HZL) software. Each accession is assigned a reference number allocated consecutively. The information contained in the record of each accession is: dataset, institution, catalogue number, scientific name, family, genus, specific epithet, taxon rank, infraspecific epithet, scientific name authorship, collection code, number of mother plants, number of tubes (in which the accession is stored), number of seeds, seed weight, community name, country, country code, state province, municipality, locality, decimal latitude, decimal longitude, geodetic datum, coordinate uncertainty in meters, elevation, collector, number of jar (in which the accession is stored), number of drawer (in which the accession is stored), collection date, sample acquisition date, % germination and observations. All data are standardized by DarwinCore 1.2 and have been validated by Darwin Test. We generated metadata with DarwinCore Archive in order to publish data in GBIF IPT. Labelling: After registering the accession in the database, seed accessions were labelled for proper identification. Scanning: Approximately 50 seeds were uniformly distributed avoiding contact between them and scanned. A resolution of 300 ppp was used, except in the case of very small seeds when 600 ppp resolution was used.
   Freezing: All of the collections in the URJC Germplasm Bank are base collections; i.e. they are stored for long periods of time and are only used in regeneration processes. Storage is carried out under optimal conditions to maximize seed viability. The procedure followed at the URJC Germplasm Bank was to store seeds in glass tubes in Kilner jars with silica gel. These jars were sealed hermetically and placed in a freezer at -18ºC.

The URJC Germplasm Bank holds the seeds of vascular plants from specific habitats. The high-mountain subcollection is composed of communities of siliceous psicroxerophilous pastures dominated by Festuca curvifolia and rich in hemicryptophytes and chamaephytes. The semi-arid subcollection is composed of gypsophytes characteristic of semi-arid environments dominated by Cistaceae, Asteraceae and Labiatae. The Lupinus subcollection is composed of seeds from different species in this genus collected in different regions in Spain. Finally, the Canary subcollection is composed of different species of genus Cheirolophus.

1. Plantae
   rank: kingdom
2. Magnoliophyta, Pinophyta
   rank: phylum
3. Liliopsida, Magnoliposida, Pinopsida
   rank: class
4. Apiales, Asparagales, Asterales, Boraginales, Brassicales, Caryophyllales, Cupressales, Ericales, Fabales, Gentianales, Lamiales, Liliales, Malpighiales, Malvales, Poales, Ranunculales, Rosales, Saxifragales
   rank: order
5. Compositae, Labiatae, Cistaceae, Gramineae, Caryophyllaceae, Cruciferae, Leguminosae, Scrophulariaceae, Campanulaceae, Plumbaginaceae, Juncaceae, Liliaceae, Ranunculaceae, Crassulaceae, Cupressaceae, Iridaceae, Linaceae, Plantaginaceae, Polygonaceae, Rosaceae, Saxifragaceae, Amaryllidaceae, Boraginaceae, Gentianaceae, Primulaceae, Umbelliferae, Violaceae
   rank: family

High-mountain pastures (psicroxerophylous pastures) and semi-arid habitats (gypsophylic steppes) in the center of the Iberian Peninsula.