Current status of the forage grass collection at Macedonian Gene Bank

Suzana Kratovalieva1, Gordana Popsimonova1, Zoran Dimov2, Sonja Ivanovska2

Institute of Agriculture, 1000 Skopje, Republic of Macedonia1 Faculty of Agricultural Science and Food, 1000 Skopje, Republic of Macedonia2

Status at national level The forage grasses collection is maintained in the gene bank placed at Institute of Agriculture-Skopje (IA) comprising 77 accessions in total. Last decade ago due to inadequate seed storage facilities and insufficient care the existed material that accounted 34 accessions of grasses are decreased and lost almost of it. Since 2004 activities on collection and conservation have been restarted under SEED Net financial support directed mainly on collecting from wild flora. A considerable grass germplasm collection including more than 100 accessions collected in 80's from Macedonian territory are stored in National Seed Storage Laboratory in Fort Collins in lack of storage facilities. Negotiations concern germplasm repatriation has started several months ago would go to be completed for one year period. Macedonia as CBD ratification country and in the face of IT PGRFA signing, more attention is paid to build gene bank through short collecting expedition throughout Macedonia as fundamental aims.

Collection The grasses collection includes 77 accessions belonging to 8 genera (Festuca spp. fescue, Dactylis spp. orchard grass, Bromus spp. brome, Koeleria spp. crested hair grass, Avena spp. oat, Briza spp. perennial quaking grass, Poa spp. bluegrass and Phleum spp. timothy) (Table 1). The biggest part of collection comprises representatives of fescue, brome following by orchard grass, crested hair grass and oat. 44 accessions only are stored according gene bank standards, while the 32 rested (F.rubra-7, D.glomerata-11, Briza media-1, Poa pratensis-1, Phleum pratense-1, A.sativa-11) are supplied with passport data and waiting to be set on field by aim of regeneration. This collection includes accession originated from Macedonia.

Table 1. Current status of the grass collection by number of accessions

Genus Species No of accessions Origin Avena Avena sativa 12 MKD Briza Briza media 1 MKD Bromus tectorum 1 MKD Bromus squarosus 3MKD Bromus sterilis 1 MKD Bromus Bromus japonicus 2 MKD Dactylis Dactylis glomerata 14 MKD Festuca pratensis 28 MKD Festuca rubra 10 MKD Festuca Festuca arundinacea 2 MKD Koeleria Koeleria macrantha 1 MKD Phleum Phleum pratense 1 MKD Poa Poa pratensis 1 MKD Total 77

Biological status of grasses collection National forage collection of grasses is completely maintained by gene bank in IA- Skopje. Concern biological status of long term conserved collection is comprises 1 advanced cultivar of Festuca arundinacea, 2 landraces from which 1 of the same species and 1 of Avena sativa, 2 have weedy status Bromus sterilis and B. tectorum; the most part originated from wild flora 39 from which 28 of Festuca pratensis, 2 of F. rubra, 3 of Dactylis glomerata, 2 of Bromus japonicus, 3 of B. squarrosus and 1 of Koeleria macrantha that account 88,64% collection (Table 2).

Table 2. Current status of the long term conserved grass collection by biological status point Genus Species Advanced Landraces Weedy Wild Total for % cultivars gene bank Avena Avena 1 1 2.27 sativa Bromus Bromus 2 4.55 japonicus Bromus 3 6.82 squarrosus 7 Bromus 1 2.27 sterilis Bromus 1 2.27 tectorum Dactylis Dactylis 3 3 6.82 glomerata Festuca Festuca 28 63.64 pratensis Festuca 1 1 4.55 arundinacea 32 Festuca 2 4.55 rubra Koeleria Koeleria 1 1 2.27 macrantha Total 1 2 2 39 44 100.00

Conservation and utilization The gene bank of IA in Skopje meets the international requirement standards (FAO/IPGRI 2006). Necessary equipment of seed preparation for long-term storage and monitoring is purchased; a seed-processing laboratory, drying cabinets, seed moisture content analyzer, an automatic seed counter, germination cabinets, thermo- refrigerator, batch sealing machine and vertical friezes. The equipment for Macedonian gene bank was provided by financial support with SEED Net project. Immediately after seed harvesting as soon as possible seed samples are cleaned, graded and moisture content determine by remark seeds which contains >12% moisture are denied. Until now seed drying is performed in drying cabinets under 24°C and air relative humidity of 12-14%, but procedure for suitable dehumidifier dryer is on going. Acceptable values of seed moisture are moving from 3.5% (oil crops), 7.0% (cereals) to 7.5% (grains) depending of crop biology. After 5-9 weeks seeds are achieved limited moisture content and packed in four-layer aluminum foil bags (10 000-12 000 seeds except 3000 seeds for large grain legumes per accession) in 5 different type of collections (Table 3). Collections are stored under -18°C differ of temporary and working maintained at +4°C. Since 2005 are re-activated plant breeding activities mainly focused on fescue, orchard grass and perennial ryegrass. Special attention is paid on collaboration and mutual seed exchange with PGR holders on national and international level in region. All accessions are available for distribution.

Table 3. Storage of grasses accessions by type of collections and distribution Number of seeds Total Safety no of Total Base Active base Viability seeds per Species stored collection collection collection Distribution test accession Avena 3000 1 000 sativa 1 5 000 (2x1 500) 600 (10x100) 400 10 000 Bromus 4 000 1200 tectorum 1 6 000 (2x2 000) 800 (10x120) 500 12 500 Bromus squarrosus, B. sterilis, 3 000 1 000 B.japonicus 3 5 000 (2x1 500) 600 (10x100) 400 10 000 Dactylis 3 000 1 000 glomerata 3 5000 (2x1500) 600 (10x100) 400 10 000 3 000 1 000 F.arundinacea 2 5 000 (2x1500) 600 (10x100) 400 10 000 Festuca pratensis, 4 000 1 200 F.rubra 28 6 000 (2x2 000) 800 (10x120) 500 12 500 Koeleria 400(2x2 1 200 macrantha 1 6 000 000) 800 (10x120) 500 12 500

Regeneration Until 2000 are being forced breeding activities on fescue and orchard grass, while the regeneration process has been carried out systematically only on few accessions. Since 2005 all accessions have been regenerated achieving standards for entry and storage in gene bank. A methodology combining spatial isolation of 30 m between regeneration plots. According gene bank procedure stored accession will be regenerated upon request from gene bank when the germinate rate in the control tests after 5 years of storage falls below 80%.

Documentation Management data (passport, characterization, gene bank management) are fully computerized. Data from inventorization are put in main database table designing according to EURISCO inventory form and are uploaded; passport data are currently available for all accessions 77 (100.00 %) and descriptive ones are entered for 14 (20.79%) from total (Table 4).

Table 4. Documentation status of grass collection Characterization/evaluation Genus Species Passport data data number % Festuca pratensis 28 - - Festuca rubra 10 - - Festuca Festuca arundinacea 2 2 2.50 Dactylis Dactylis glomerata 14 3 3.75 Bromus tectorum 1 1 1.25 Bromus squarosus 3 3 3.75 Bromus sterilis 1 1 1.25 Bromus Bromus japonicus 2 2 2.50 Avena Avena sativa 12 1 1.25 Koeleria Koeleria macrantha 1 1 1.25 Briza Briza media 1 Poa Poa pratensis 1 Phleum Phleum pratense 1 Total 77 14 20.79

Identification of the Most Original Sample (MOSs) The MOSs of database has been updated (Table 5). In the forage database there are 94 accessions in total from which 92 (97.87%) are declared MOS-1 and 2 (2.13%) MOS- 4 (more away). For grasses provisional identification of the originality of 3 genera (orchard grass, fescue, timothy, bluegrass) 56 accessions (100.00%) are identified MOS-1 until legume collection is completed by 38 (94.74%) MOS-1 (alfalfa) and 2 (5.26%) MOS-4 (clover).

Table 5. Identification of the MOSs No of accessions Genus Species Total ORIGCTY Originality 1 2 (with 3 (one 4 5 (MOS) MOS) away) (more (unknown) away) Priority genera Dactylis 14 MKD 14 - - - - D. glomerata 14 MKD 14 - - - - other Dactylis spp. ------Festuca 40 MKD 40 - - - - F. pratensis 28 MKD 28 - - - - F. rubra sensu lato 10 MKD 10 - - - - F. arundinacea 2 MKD 2 - - - - other Festuca spp. - MKD - - - - - Lolium ------L. perenne ------L. multiflorum ------L x hybridum (boucheanum) ------other Lolium spp. ------Medicago 2 MKD 2 - - - - M. sativa 2 MKD 2 - - - - other perennial Medicago spp. ------annual Medicago spp. ------Phleum 1 MKD 1 - - - - Ph. pratense 1 MKD 1 - - - - other Phleum spp. ------Poa 1 MKD 1 - - - - P. pratensis 1 MKD 1 - - - - other Poa spp. ------Trifolium 36 MKD 34 - - 2 - T. pratense 24 MKD, SVN 23 - - 1 - T. repens 1 SVN - - 1 - other Trifolium spp. 11 MKD 11 - - - - Total 94 92 - - 2 -

Collecting activities Collecting activities organized within the framework of SEED Net project 2007 are carried out in collaboration with Slovenian partner. Collecting mission was performed in east part of Macedonia in meadows and grasslands by particular interest directed to meadow fescue and red clover. All sites visited during collecting mission were mainly chosen during discussion with local people who well know the region and agricultural practice. A total of 95 forage accessions collected in 31 natural habitats 49 are fall down on grasses. Accessions were collected as seeds and passport data are yet prepared for each accession. Geographical coordinates of each site were recorded using GARMIN 12 GPS receiver.