Hojka Kraigher CONSERVATION OF FOREST GENETIC RESOURCES WITH FOREST REPRODUCTIVE MATERIAL MANAGEMENT GUIDELINES Maribor, January 2025 Title CONSERVATION OF FOREST GENETIC RESOURCES WITH FOREST REPRODUCTIVE MATERIAL MANAGEMENT GUIDELINES Author Hojka Kraigher (Slovenian Forestry Institute) Review Franc Batič (University of Ljubljana, Biotechnical Faculty) Mitja Kaligarič (Univerza v Mariboru, Faculty of Natural Sciences and Mathematics) Translation Miha Odar, Franc Batič, Hojka Kraigher Native speaker corrections Jean McCollister Technical editors Katja Kavčič Sonnenschein (Slovenian Forestry Institute) Jan Perša (University of Maribor, University Press) Graphics material Illustrators: Anja Rupar, Eva Margon, Klara Jager, Marija Prelog, Marina Gabor, Metka Kladnik, Teja Milavec. Illustrations are used with the permission of the authors. Photos by: Melita Hrenko, Hojka Kraigher. Own sources unless otherwise stated. Kraigher (author), 2025 FDC classification Maja Peteh (Slovenian Forestry Institute) Design and page break Lenka Trdina (Dvokotnik, Lenka Trdina, s. p.) and Metka Slamič (Š.K.R.A.T. Boštjan Žuran s.p.) Cover drawing Author: Eva Margon, 2024 One-year oak sapling Published by University of Maribor, University Press, 2025 Slomškov trg 15, 2000 Maribor, Slovenia https://press.um.si, zalozba@um.si Issued by Univerza v Mariboru, Faculty of Natural Sciences and Mathematics, Koroška cesta 160, 2000 Maribor, Slovenia https://fnm.um.si, fnm@um.si Edition First edition, translation of the original work from the Slovenian language. Kraigher, H. (2024). Ohranjanje gozdnih genskih virov s semenarskim praktikumom. Univerza v Mariboru. Univerzitetna založba. doi: 10.18690/um.fnm.3.2024 Published at Maribor, Slovenia, January 2025 Publication type E-book Available at https://press.um.si/index.php/ump/catalog/book/942 This publication is co-financed by the Public Agency for Scientific Research and Innovation Activities of the Republic CIP - Kataložni zapis o publikaciji of Slovenia (ARIS), the Ministry of Agriculture, Forestry and Food of the Republic of Slovenia, the Ministry of the Univerzitetna knjižnica Maribor Environment, Climate and Energy, Slovenian Forestry Institute, LIFEGENMON LIFE12 ENV/SI/000148, LIFE SySTEMiC 630*2:631.53.02(075.8)(0.034.2) LIFE 18 ENV/IT/000124, ARIS Research Programme Forest Biology, Ecology and Technology (P4-0107) and several ARIS KRAIGHER, Hojka targeted research programmes (V4 2222, V4 2015, V4 1438, V4 1616). The LIFEEGENMON and LIFE SySTEMiC projects Conservation of forest genetic resources with were funded under the LIFE Programme, the EU financial instrument for environment and climate action. forest reproductive material management guidelines [Elektronski vir] / Hojka Kraigher. - 1st ed. - E-publikacija. - Maribor : University of Maribor, University Press, 2025 Način dostopa (URL): https://press.um.si/index.php/ ump/catalog/book/942 ISBN 978-961-286-949-6 Co-funders: doi: 10.18690/um.fnm.2.2025 COBISS.SI-ID 223549699 REPUBLIC OF SLOVENIA MINISTRY OF NATURAL RESOURCES AND SPATIAL PLANNING © Univerza v Mariboru, Univerzitetna založba / University of Maribor, University Press Besedilo / Text © Kraigher (author), 2024 To delo je objavljeno pod licenco Creative Commons Priznanje avtorstva-Deljenje pod enakimi pogoji 4.0 Mednarodna. / This work is licensed under the Creative Commons Attribution-ShareAlike 4.0 International License. Users are allowed to reproduce, distribute, lease, publicly perform and adapt the copyright work, provided that they credit the author and redistribute the work/adaptation under the same conditions. Commercial use is also permitted for new works resulting from the adaptation. All third-party material in this book is published under a Creative Commons licence unless otherwise stated. If you wish to reuse third-party material that is not covered by a Creative Commons licence, you will need to obtain permission directly from the copyright holder. https://creativecommons.org/licenses/by-sa/4.0/ ISBN 978-961-286-949-6 (pdf) 978-961-286-950-2 (softback) DOI https://doi.org/10.18690/um.fnm.2.2025 Price Free copy For publisher Prof. Dr. Zdravko Kačič, Rector of the University of Maribor Attribution Maribor Kraigher, H. (2024). Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines. University of Maribor, University Press. doi.org/10.18690/um.fnm.2.2025 TABLE OF CONTENTS PREFACE .............................................................................................................................................................................................................................1 1 INTRODUCTION .................................................................................................................................................................................................................... 3 1.1 Conservation of Forest Genetic Resources and the SIFORGEN Programme ................................................................................ 4 1.2 Phenology ....................................................................................................................................................................................................................6 2 FROM SEED TO SEEDLING ................................................................................................................................................................................................7 2.1 Flower, Seed and Fruit ..........................................................................................................................................................................................8 2.2 Seed and Seedling Growth and Development .........................................................................................................................................10 2.2.1 Anatomy and Chemical Composition of Woody Plant Seeds ............................................................................................11 2.2.2 Germination ................................................................................................................................................................................................11 2.2.3 Germination Physiology ....................................................................................................................................................................... 13 2.2.4 Seed aging ................................................................................................................................................................................................... 14 2.2.5 Seed Dormancy ......................................................................................................................................................................................... 14 2.2.6 Seed Quality Analyses ...........................................................................................................................................................................16 2.2.7 Emergence of Seedlings .......................................................................................................................................................................16 2.3 Tree Nursery Basics ...............................................................................................................................................................................................18 3 THE PHYSIOLOGY OF FLOWERING AND SEED PRODUCTION IN FOREST TREES .................................................................................. 19 3.1 Flowering Periodicity ............................................................................................................................................................................................20 3.2 Reproductive Cycles ...............................................................................................................................................................................................21 3.3 Initiation and Stimulation of Flowering .......................................................................................................................................................21 4 BASICS OF FOREST GENETICS .....................................................................................................................................................................................25 4.1 Mendel’s and Morgan’s Laws ............................................................................................................................................................................26 4.2 Population Genetics, Evolution and Speciation ..................................................................................................................................... 27 5 FOREST REPRODUCTIVE MATERIAL PRODUCTION AND TREE NURSERY TECHNOLOGY .......................................................................31 5.1 Fructification in Forest Tree Species .............................................................................................................................................................32 5.2 Collecting, Cleaning and Sorting of Seeds .................................................................................................................................................33 5.3 Seed Drying ...............................................................................................................................................................................................................35 5.4 Seed Storage .............................................................................................................................................................................................................36 5.5 Work Procedures for Seedling Cultivation in Nurseries ......................................................................................................................38 5.6 Container Seedling Cultivation ........................................................................................................................................................................39 5.7 Mycorrhizal Seedling Cultivation ....................................................................................................................................................................39 5.8 Effects of Nursery Practice on Genetic Diversity – from Stand to Seedling, Genetic Diversity Decreases.................40 6 FOREST REPRODUCTIVE MATERIAL ACT ...................................................................................................................................................................43 6.1 Interpretation of Terminology According to the Forest Reproductive Material Act (2002) ................................................45 6.2 Minimum Requirements for the Production of “Source Identified” Forest Reproductive Material............................... 47 6.3 Minimum Requirements for “Selected” Seed Stands........................................................................................................................... 47 6.4 Forest Seed Facilities for the Production of the “Qualified” and “Tested” Categories of FRM ........................................48 6.5 Production of Forest Reproductive Material .............................................................................................................................................48 6.6 Master Certificate for Forest Reproductive Material .............................................................................................................................50 6.7 Forest Reproductive Material Lot ...................................................................................................................................................................50 6.8 Reserves of Forest Reproductive Material ..................................................................................................................................................51 6.9 Comparison of the Competences of National Legislation and EU requirements ..................................................................52 6.10 Meaning, Competences and Sequence of the Rules .............................................................................................................................53 7 REGULATORY PROVISIONS CONCERNING FOREST REPRODUCTIVE MATERIAL .......................................................................................55 7.1 Demarcation of Regions of Provenance ......................................................................................................................................................56 7.1.1 Seed Districts (1951-1986) ...................................................................................................................................................................................56 7.1.2 Seed Units (1986-2002) ........................................................................................................................................................................................ 57 7.1.3 Demarcation of Slovenia into Regions of Provenances (2002-) ...................................................................................................... 57 7.2 List of Species to Which the Forest Reproductive Material Act (ZGRM) Applies .....................................................................59 7.3 Procedures for the Approval of Forest Seed Facilities .........................................................................................................................60 7.4 FRM Certification Procedures ...........................................................................................................................................................................60 7.5 Overview of the Series of Procedures for Approval of FSF and FRM Certification ................................................................. 61 8 FOREST SEED FACILITIES ................................................................................................................................................................................................63 8.1 Seed trees or stands for the “source identified” FRM category ......................................................................................................64 8.2 "Selected" Seed Stands .......................................................................................................................................................................................64 8.2.1 Description and Tending of Selected Seed Stands ................................................................................................................65 8.2.2 Some Difficulties in the Selection and Tending of Selected Seed Stands ..................................................................67 8.3 Organisation of Seed and Seedling Supply ..............................................................................................................................................68 8.4 Systemic Problems in Forest Reproductive Material Production and Tree Nursery Activities (2017 Summary) .....70 9 SEED AND SEEDLING DATA FOR SELECTED TREE SPECIES .............................................................................................................................. 73 9.1 European Beech (Fagus sylvatica L.) ............................................................................................................................................................ 74 9.1.1 Flowering and Fructification............................................................................................................................................................... 75 9.1.2 Harvesting and Processing of beechnuts .................................................................................................................................... 76 9.1.3 Storage of Beechnuts, Dormancy Breaking and Germination ........................................................................................... 76 9.2 Oaks: Pedunculate Oak (Quercus robur L.) and Sessile Oak (Quercus petraea Liebl.) ........................................................78 9.3 European ash (Fraxinus excelsior L.) ........................................................................................................................................................... 81 9.4 Norway Maple (Acer platanoides L.) ...........................................................................................................................................................83 9.5 Sycamore (Acer pseudoplatanus L.) ..............................................................................................................................................................84 9.6 Black Alder (Alnus glutinosa (L.) Gaertn.) ..................................................................................................................................................86 9.7 Wild Cherry (Prunus avium L.)..........................................................................................................................................................................88 9.8 Selected Conifers....................................................................................................................................................................................................90 9.8.1 European Silver Fir (Abies alba Mill.) .............................................................................................................................................90 9.8.2 European Larch (Larix decidua Mill.) ..............................................................................................................................................90 9.8.3 Norway Spruce (Picea abies Karst) ................................................................................................................................................. 91 9.8.4 Pines (Pinus spp.) .................................................................................................................................................................................... 91 9.8.5 Douglas Fir (Pseudotsuga menziesii (Mirb.) Franco) ..............................................................................................................92 10 REFERENCES ........................................................................................................................................................................................................................95 10.1 Textbooks, Protocols and Thematic Monographs ....................................................................................................................................96 10.2 Other References ....................................................................................................................................................................................................96 10.3 Overview of Slovenian Legislation Relating to FRM ............................................................................................................................98 11 TABLE OF ANNEXES .........................................................................................................................................................................................................99 Annex 1: Protocol for the management of forest tree seedlings from the time they are extracted from the nursery to the time they are planted, in cases where seedlings are provided by the Slovenian Forest Service from the Slovenian budget funding .............................................................................................................. 101 Annex 2: Documents for the approval of in situ seed facilities .........................................................................................................104 Annex 3: Reporting form for the harvesting of forest reproductive material for the purpose of obtaining a master certificate ...............................................................................................................................................................................109 Annex 4: Master Certificate of Forest Reproductive Material (Specimen) .....................................................................................110 Annex 5: The certificate of the Inspectorate of Agriculture, Forestry, Hunting and Fishing (IRSKGLR) on the mixing of forest reproductive material ........................................................................................................................................111 Annex 6: Seed Quality Certificate issued by the Slovenian Forestry Institute .............................................................................112 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines PREFACE Large-scale disturbances, such as extreme weather, wildfires, and pests, have weakened forests across Europe, reducing their ability to regenerate and adapt to climate change. To address this, professional seed and nursery manage-ment practices are essential to maintain forest health and resilience. This textbook and included guidelines were developed with the Slovenian environment in mind but are appli-cable across Europe. Since 2000, we have emphasized the importance of forest reproductive material and tree nursery activities for successful, close-to-nature, and multifunctional forest management, which forms a cornerstone of forest management practices. Following 2001, long-lasting droughts became more frequent, and in 2014, glaze ice affected 40% of Slovenian forests. This accelerated bark beetle infestations, while already weakened forests were further damaged by severe windthrows in the subsequent years. Forests affected by such disturbances can recover naturally, but restoring commercially viable forests is a lengthy process. During this recovery, issues like weed infestation and overpopulation of herbivorous wildlife often arise. Large-s-cale disturbances have also impaired the natural regeneration and genetic diversity of young forest stands, making them less adaptable to future environmental changes. This highlights the growing recognition of the need for professional seed and nursery management, based on local knowledge and forest nursery capacities. Although natural regeneration remains common in close-to-nature mana-ged forests, it is no longer the primary method of regeneration. A shift in strategies, planning, and support for this critical aspect of forestry is necessary to address these emerging challenges. Large-scale disturbances have also affected the natural regeneration capacity and genetic diversity of natural young forest stands, which will be even less able to adapt to a changing environment in the coming decades. This is why awareness of the importance of professionally appropriate seed and nursery production, based on local knowledge and capacities of forest tree nurseries in the Slovenian territory, is returning to the forestry sector. Natural regeneration still prevails in our close-to-nature managed forests, but it is no longer the main regeneration practice in the Slovenian Fo-restry School; what is needed is a change in thinking, strategies, planning and support for this long-neglected sector of forestry in Slovenia. Within the context of several targeted developmental projects (CRP), we have set ourselves the task of prepa-ring forest reproductive material management guidelines with a focus on the conservation of forest genetic resources as a basis for the sustainable management of multifunctional forests in times of rapid environmental change, increased demand for the use of forest reproductive material and for maintaining the adaptive potential of future populations of forest trees to a changing environment. Consequently, conserving the genetic diversity of forest trees is in line with the Slovenian Forest Genetic Resources Programme (SIFORGEN), which is also part of the European Forest Genetic Resources Programme (EUFORGEN). The draft of the Seed Practicum – Forest Reproductive Material Management Guidelines was distributed as wor-king material (in five spiral-bound copies) at the Public Forestry Service workshop for the needs of the Slovenia Forest Service and the forest seed and nursery sector in 2019. I would hereby like to thank Professor Emeritus Franc Batič and Academic Ivan Kreft for providing peer reviews of this draft. However, it has taken five years to complete and finalise the material, to accompany it with original illustrations, and to propose it to the University of Maribor for review as a textbook entitled Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines (Seed Pra-cticum); Prof. E. Franc Batič, retired professor of botany and plant ecology, and Prof. Dr. Mitja Kaligarič, full professor of botany at the University of Maribor, were nominated as reviewers. I take pride in quoting the reviewers: “The work comprehensively presents all the elements relating to forest genetic resources. It gives a historical overview of the issue and its management in the EU and Slovenia. The biological and genetic basis of forest genetic reso-urces, silvicultural measures for the selection and management of forest seed resources, as well as the related essential basics concerning forest seed husbandry and nursery activities, are presented in an appropriate manner. 1 Preface /…/ I consider this work to be of high priority, particularly at a time of great environmental change, which is not sparing our forests. For the first time in a long period, the state of forest resource management in our country and in the EU is described and collected in one place, with all the professional, technological and management basics. As such, the work will serve as an indispensable resource for everyone interested in sustainable forest utilisation (forest owners, the Slovenia Forest Service (SFS), the Chamber of Commerce and Industry of Slovenia (CCIS), the Ministry of Agriculture, Forestry and Food of the Republic of Slovenia, students, teachers and researchers in the field of forestry, etc.), as well as for all other parties interested in nature and environmental issues.” /…/ Franc Batič, Professor Emeritus, retired full professor of Botany and Plant Ecology “A highly complex subject matter is covered here, ranging from the basics of morphology, germination, genetics, forestry and legislation. There is also an emphasis on the applied aspect (“Seed Practicum – Forest Reproductive Material Management Guidelines”) - the management of forest seed resources, including forest seed and nursery activities. /…/ Therefore, the Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guide-lines (Seed Practicum) certainly covers more areas than its title promises, as it deals comprehensively with all aspects of forest genetic resources, while providing a useful recapitulation of the biological and forestry basics. Almost two-thirds of Slovenia’s national territory is covered by forest (including overgrown farmland), which makes the topic relevant and topical, including in the light of major climate changes (droughts and bark beetle infestation) and land use changes (reclamation of farmland and grazing meadows). This monograph is intended not only for foresters and forest professio-nals, but also for students of forestry, biology and ecology (the latter courses are also taught at the University of Maribor), as well as anyone who is in any way interested in forests, their management and conservation.” Prof. Dr. Mitja Kaligarič, Full Professor of Botany at the University of Maribor I would hereby also like to thank all the contributors to this textbook: I would like to thank my colleagues at the Slovenian Forestry Institute (SFI): Dr. Marjana Westergren, Dr. Gregor Božič and Dr. Peter Železnik for discussions, for the transmission of our commonly produced guidelines for seeds and seedling manipulation thanks go to Andrej Breznikar. MSc, from the Slovenia Forest Service (SFS), and to Marina Herman Planinšek and Vlado Planinšek from the Omorika Nursery for their nursery practices overview. We have also used our own workshop materials and the Forest Genetic Monitoring Handbook, which was developed within the framework of the LIFE for European Forest Genetic Monitoring System (LIFEEGENMON) project (2014-2020), which is partly based on the EUFORGEN Programme joint reports, particularly regarding the issues of genetic diversity conservation in forest seed and tree nursery production (Gömöry et al. 2021), and on the charts developed within the framework of the CRP, LIFE SySTEMiC (2019-2024) and other projects. Thanks also go to colleagues at the Department of Forest Physiology and Genetics at the Slovenian Forestry In-stitute (GIS), who contributed photographs; special thanks go to Melita Hrenko, to everyone who participated in the design and selection of the drawings, especially Katja Kavčič Sonnenschein, MSc, and to all authors of the drawings, to Dr. Maja Peteh for her help in editing the references and classifying the work, the Slovenian language proofreader Teja Kačar, the layout designer Lenka Trdina, and the editor of the Forestry Journal for permission to publish the abstract of the Systemic Problems of Reforestation meeting (from 2017). Special thanks go to our co-funders: the Ministry of Agriculture, Forestry and Food of the Republic of Slovenia, the Slovenian Agency for Scientific Research and Innovation (ARIS), the Ministry of the Environment, Climate and Energy, the Slovenian Forestry Institute, the LIFE Programme for co-financing LIFE projects, targeted research projects (V4-1616, V4-1819, V4-2015, V4-2222), the research programme P4-0107 and the Public Forestry Service (JGS). INTRODUCTION 1 Introduction 1.1 Conservation of Forest Genetic Resources and the SIFORGEN Programme Forest trees are characterised by their size, long trees to changing environmental conditions. Therefore, the lifespan and, in most cases, large genetic base, as domesti- primary task of conserving forest genetic resources (FGR) is cation and cultivation processes are relatively new, starting precisely to preserve as much genetic diversity as possible mostly in the 1950s (Nanson 2004). In natural populations in all forest governance and management practices, in par- of forest trees, genetic variability within populations is ge- ticular in terms of silvicultural measures, which should be nerally higher than between populations, and it is this high based on “genetic forest protection”, i.e. forest protection variability within the populations that allows them to main- based on the conservation of genetic diversity. Measures for tain the adaptive capacity (of future generations) of forest the conservation of forest genetic resources include: ा Silvicultural measures, which take into account the importance and potential for the conservation of genetic diversity of the populations concerned, i.e. forest tree stands and plantations. ा Silvicultural measures in support of the minority species and populations of forest trees at the edge of their area of distri- bution, and in support of particular populations of forest trees that need support for long-term survival in a particular site. ा Reforestation measures, which include: ◊ Natural regeneration of forests at times of abundant production, in populations with an adequate number of mother trees – seed trees with appropriately developed reproductive parts of their crowns. ◊ Regeneration by planting and sowing in areas: • where natural reforestation is inadequate in terms of species and genetic diversity; or • where it fails to meet the objectives of sustainable forest development in an optimal time-scale; or • where large areas of forests have been cleared as a result of large-scale disturbances (fires, windthrows, glaze ice, etc.), sanitary measures due to infestation of pests and diseases, or due to inadequately implemented felling operations; or • where the reproductive parts of crowns of trees have been damaged by past disturbances (e.g. glaze ice or snow break), reducing the genetic diversity of naturally occurring young forest regeneration centres; or • where spatial plans define a change of land use to forest plantations and forest. ◊ Regeneration by enrichment planting and sowing: • where the quality of natural saplings is not sufficient to achieve the long-term objectives of conserving forest genetic resources and the sustainable/viable development of multifunctional forests; or • where the aim is to increase the species and genetic diversity of existing young forest stands by enrichment plan- ting and sowing; or • where we want to include additional provenances of individual forest tree species as a pre-preparation of forests for (un)expected climate change. Conservation of the FGR based on the imple- mentation of the above measures, the dissemination of SIFORGEN is the Slovenian Forest Genetic knowledge on the importance of genetic diversity for the Resources Programme. today, communication with stakeholders at different Is the European Forest Genetic Resources Programme, which has been operating sustainable development of forests as we know them EUFORGEN measures for the conservation of the FGR of individual adop ted by the Ministerial Conferences for the Protection of Forests in Europe (now forest tree species and tracing the effectiveness of the levels, with the target and the general public, and concrete since 1994 on the basis of resolutions Forest Europe). Slovenia has been a parti- implemented measures through the establishment and cipant since the first meeting of national coordinators in 1995. implementation of the genetic monitoring of forests are 4 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines the basis of the Slovenian Forest Genetic Resources on forest reproductive material (FRM) in Slovenia, Programme, or SIFORGEN for short. which has been in place since 2002, when the Forest These measures are based on international Reproductive Material Act (ZGRM 2002), was adopted, strategies and are formulated jointly by with the European the European Directive on the marketing of forest Forest Genetic Resources Programme, EUFORGEN, of reproductive material (EC/105/1999) was harmonised, which Slovenia has been a member since its inception: and the list of approved forest seed facilities (FSFs, a report on the Slovenian Forestry School and the forest which, after approval, are permitted for producing FRM genetic resources in Slovenia was already presented at of different categories) is also published annually in the the first Steering Committee (SC) meeting of EUFORGEN European FOREMATIS database (http://ec.europa.eu/ in Sopron, Hungary, in 1995, and Slovenia formally signed forematis/). Since 2018, we have been participating in the membership agreement in 1997. The country also the meetings of the OECD Working Group on FRM, and participated in the first European project to organise a are a member of the Working Group on Forest Seeds and joint European database on Dynamic Conservation Units Propagating Materials since 2022, and we have previously (GCUs), EUFGIS, and in several working groups during all attended the regular annual meetings of the European the phases of the EUFORGEN Programme, which entered Working Group on Forest Reproductive Material within DG its sixth operation phase in 2020. Experts and the face-to- SANTE. Since Slovenia's accession to the EU, the Ministry face meetings within the context of the programme also has been sending a representative to the regular annual contributed to the effective design of the new legislation meetings of the Working Group in Brussels or Paris. Photo 1: Mast beechnut production: beechnuts hang from all the branches throughout the crown (photo by Hojka Kraigher). 5 Introduction 1.2 Phenology Usually, forest trees bloom in the year after flower PHENOLOGY stem induction and mostly from April to May, in many species defines the growth and development of forest trees. before the start of leafing and the growth of vegetative shoots, which facilitates pollen dissemination and pollination. Early The growth and development of forest trees flowering also makes them more susceptible to late spring is significantly defined by phenology – the onset of the frosts. In a given forest tree population, the onset of flowering developmental stages within the context of the vegetative of different trees may extend over several weeks, and there growth-development cycle and of the generative growth- may also be differences in different parts of the crown of the development cycle, as well as lignification and senescence, same tree. Self-pollination is reduced because of the different which have an important impact on the winter survival of maturation times of male (protandry – male flowers mature first) woody plants in temperate climates. and female flowers (protogyny – female flowers mature first). In the warm temperate climate zone of the northern Flowering leads to pollination either by wind (anemophily) or hemisphere, the year starts with a dormancy phase in Janu- insects (entomophily), which takes place over one to two weeks ary and February, mainly conditioned by low temperatures in dry weather, interrupted by rainfall. In most tree species, and, in the case of some alien species, a short photoperiod. pollination is immediately followed by fertilisation, though In March (equinox), the dormancy is no longer affected by the with others it starts the year after pollination (pines and oaks) short photoperiod, while in April and May vegetative shoots or in the third year after pollination (cedars). The fruits and start to grow and develop, which depends on the increase cones usually reach their final size in July, followed by a period in ambient temperature. This is also the period when height of maturation that most species reach between September and (axial) and thickness (radial) growth begins, which ends in December. This is followed by the dispersal and dissemination July to August for most tree species. Height growth ends in of the seed, usually in the autumn, though it can last until the the summer months, between late June (pines) and late Sep- following spring. tember (larch), except in individual species where favourable Mature seeds can be dormant (e.g. larch, ash and conditions (humidity or fertilisation) after the end of growth Douglas fir), and breaking of dormancy requires stratification, may stimulate further growth in August. However, for most i.e. a period of exposure to moisture and cold, or in some species, thickness growth ends in September. Although the species a period of alternating cold and warm periods shortening of the photoperiod (autumnal equinox) affects (warm, cold or alternating stratification), or the mechanical the onset of dormancy in a different manner depending on or chemical treatment of the seed – scarification. species and ecotype, it includes bud development, an increa- Forest tree species can be monoecious where the se in osmotic pressure and dry matter content, the cessation female and male flowers grow on the same plant (flowers of stem growth and lignification, and the storage of nutrients. may also be hermaphroditic) or dioecious where only male Development of generative organs begins with or only female flowers are present on some individual speci- the induction of flower stems in the year before flowering, mens. There are also transitions between the two forms, and in June to early July for most species (Wareing 1958, in monoecic and diecic forms can appear in the same species, Nanson 2004). This is stimulated by a warm period of dry or even triecious trees with hermaphrodite flowers, e.g. in ash and sunny days. In addition, the trees must have sufficient trees; dioecious male trees usually grow better than female reserve substances that can be used at the time of flowering trees because all the assimilates are used for the growth of (therefore, for example, oaks and beeches do not form flower the tree alone and not for the production of the seeds. stems if there was a high mast production the previous year). In the following sections, we look at the flower, The induction of flower stems can be influenced by a variety seed and fruit, as well as the flowering and mast production of factors, ranging from hormonal regulation in natural processes in more detail. systems (including interactions with symbiont organisms) to the stimulation of flowering in seed plantations, e.g. by There are monoecious, dioecious and triecious tree inducing stress (drought stress or the removal of part of the species. crown tissue) or by the use of plant hormones. 6 TO SEEDLING FROM SEED 2 From Seed to Seedling 2.1 Flower, Seed and Fruit The contents are mainly taken from Mala flora Slovenije (Little Flora of Slovenia) (2007). A flower is a short, rosette-type, limited-growth be much more complex within this species). The flowers of shoot with transformed leaves for sexual reproduction. gymnosperms are mostly unisexual, the plants are mostly In the case of hermaphrodite flowers the stamens and monoecious. pistil(s) develop in the same flower. Unisexual flowers only The transfer of the pollen grain to the stigma of have stamens or only pistil(s) developed. Plants with male the pistil of angiosperms and to the ovule of gymnosperms and female flowers on the same plant are monoclinous or is called pollination. Self-pollination (autogamy) is monoecious (hazels, black alders, spruces, firs, oaks, beeches pollination with pollen grains from the same genotype. and hornbeams). Plants that only have female flowers on Allogamy is pollination by external pollen, zoophily is the one plant and male flowers on the other are diclinous transfer of pollen grains by insects, and anemophily is the or dioecious (hops, yews, willows, poplars and junipers). transfer of pollen grains by the wind. Plants whose populations only have unisexual (either The pollen grain on the stigma of the pistil gynoecious or androecious), female and hermaphrodite, germinates and grows directly to the ovules. After male and hermaphrodite or only hermaphrodite flowers fertilisation, the ovule starts to develop into a seed. The on the same specimens are polygamous (such as horse formation of an embryo without fertilisation is called chestnut). Triecious plants have only female, only male or agamospermy. In the case of angiosperms, the seed only hermaphroditic flowers on individual specimens (Little develops in the ovary, while in the case of gymnosperms, Flora provides the example of the European ash, but the the seed develops on the surface of the carpels. occurrence of different sex combinations of flowers can Parthenocarpy is the development of fruit without seeds. A seed is a young plant, a living organism in which not have it, the spare nutrients are stored in the cotyledons, metabolic processes are slowed down or suppressed. The which fill most of the space in the seed. seed is enclosed in a seed coat (testa). This is particularly Important concepts in forest seed production include: strong in the case of seeds that do not remain in the fruit. • embryo (consisting of the radicle, plumule and The hilum is the part of the seed where the funiculus cotyledons), (funiculus cord) was attached, by which the seed was • endosperm (nutrient reserve, often very thin or connected to the placenta in the ovary (the carpel). Inside non-existent), the seed is the initial stage of the developing plant germ • seed coat. (embryo) and often also the nutritive tissue (endosperm). Depending on the method of fertilisation, the endosperm SEED is haploid (gymnosperms), diploid or triploid (secondary is a young plant, a living organism in which meta-bolic processes are slowed down or suppressed. endosperm). Conifer seeds have an endosperm, while the seeds of our deciduous trees often do not. If a seed does After fertilisation, the pistil, and sometimes also develop from the entire inflorescence – a large number of some other parts of the flower (insertion point, bracts, blossoms (mulberry, fig, etc.). The fruit is succulent (fleshy) etc.) develop into the fruit (i.e. a pericarp), which consists when the pericarp is fully or partially fleshy and dry when of an exocarp, mesocarp and an endocarp, which could be the pericarp is dry. A dehiscent fruit opens for the seeds to quite different in structure). The fruit contains seed and it be released, while a closed fruit (indehiscent) falls off as a develops only in angiosperms. This can be simple, when whole (achenes). The fruits are extremely diverse in their it is formed mainly from the ovary of pistil (beechnuts, formation and structure. acorns, samaras of maple, elm, hornbeam, linden trees, etc.), or drupelets (berries), when it is formed mainly from FRUIT the receptacle, on which there are several pistils (e.g. apple, consists of seed(s) and pericarp. raspberry and strawberry fruits, etc.); composite fruits 8 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines 1) Simple fruits develop from one or more mature pistils. ा Simple fleshy fruits. ा Fleshy indehiscent fruits: fall off complete when ripe. Berries: the fruit is fully fleshy and produced from one (barberry) or several carpels (bilberry). Drupaceous (stony) fruits: only the outer part of the pericarp (exocarp and mesocarp) is fleshy and the inner part is woody (stone – endocarp); they are formed from one (cherry) or several mature carpels (walnut, elder and olive). ा Fleshy dehiscent fruits: are fleshy capsules (spindle tree – Euonymus sp., touch-me-not - Impatiens sp.). ा Simple dry fruits. ा Dehiscent dry fruits: at maturity, the fruits open and the seed is shaken out. The follicle develops from a single carpel. When ripe, it opens along the abdominal suture (hellebore, larkspur (Delphinium sp.) and peony). The pod is a specially shaped follicle, and when ripe it opens along the abdominal and dorsal sutures (pea, bean, golden chain (Laburnum sp.) and black locust). The capsule develops from a pistil, which is made up of two or multiple carpels, with either a single or multiple compartments. When ripe, it opens with elongated flaps (horse chestnut); or with calyx teeth at the top (primrose) or with the cap falling off (black henbane); or the seeds shoot out through holes (poppy). The siliqua is a distinctive capsule that develops from two carpels. If the siliqua is less than three times as long as it is wide, it is a silicle. When ripe, it opens in two flaps and the seeds disperse from the central partition or septatum (crucifer). ा Indehiscent dry fruits: their pericarp does not open and the seed is therefore not disseminated alone but together with the pericarp. The nut is a single-seeded fruit with a dry pericarp. Syncarpous nuts (from two or more carpels) may be wing nuts or samaras (maple, ash, birch, alder and elm) or have no wings (hazelnut, beechnut, acorn, buckwheat and hemp nuts). Nut-like form of the fruit is also the caryopsis of some cultivated grasses (wheat, rye and maize) and the glumaceous fruit of many grasses and some grains (oats and barley), and the achene of composites (sunflower and dandelion), the seed in caryopsisis grown with the seed coat but not in achene. In the case of a glumaceous fruit, the fruit is accompanied by the palea and sometimes also the lemma. Schizocarps or splitting fruits are fruits that can split into as many fruitlets as there are carpels (umbelliferous plants and maple). The fruitlets are often connected by a petiole (the carpophore in umbelliferous plants) or have appendages that aid in their dispersal (maple seed wings). Some other schizocarpic fruit are multi-seeded closed fruit that breaks into single-seeded fruitlets. These include the loment – consisting of a single carpel that breaks transversely into single-seeded fruitlets (crownvetch), the lomentose siliqua – consisting of two or four carpels that break transversely (radish), and the schizocarpic nutlet (labiate and boraginaceous plants), which breaks into four single-seeded fruitlets at maturity. 2) Aggregate fruits develop from flowers with seve- considered as composite fruits (with the caveat that the ral separate carpels that mature into individual fru- seeds develop on the surface of the carpels) but as the itlets aggregated in fruit which is spread as a whole pistil is not developed they are not proper fruit. Spruce, (raspberry, blackberry, apple) or they fall off separately pine and fir cones have woody seed scales, while juniper (buttercup, cinquefoils). The fruitlets can be drupaceo- berries have fleshy scales. us (raspberry or blackberry) and apple (apple or pear tree). Aggregate nutlets may be fully dry (buttercup and DISSEMINATION OF SEEDS: be either on the fleshy receptacle (strawberry) or insi- dislodging seeds and fruit. • cinquefoil) or partially fleshy, in which case they may • Autochory – the plant has a mechanism for de it (rosehip). external factors (among types of allochory: Allochory – is carried out with the help of 3) Composite fruits develop from inflorescences that water - hydrochory, wind - anemochory, give the appearance of a single fruit (fig, mulberry, animals and man - zoochory). pineapple and hop strobiles). Conifer cones are also 9 From Seed to Seedling 2.2 Seed and Seedling Growth and Development Growth and development are how plants grow in response of a plant, the result of interactions between size and develop different shapes and patterns of leaves, genetic composition and the environment that are based flowers and roots. This consists of: on physiological processes during growth. The heredity of ा Morphogenesis – the development of cell and plants is mainly stored in DNA in chromosomes, which re- organ shape. It depends mainly on the regulation gulates the synthesis of proteins and enzymes providing of the direction of cellular enlargement and the feedback control of the structure of cells and the response control of the plane of cell division. of plants through transfer RNA. Genetic variability in plant ा Differentiation – the process by which cells un- growth and development can be caused by gene mutati- dergo biochemical and structural changes for the ons, recombination and gene migration. performance of certain functions. The daughter properties that are different from the parent cell. sponses of a plant, the result of interactions betwe- en heredity and the environment that are based on cell attains metabolic, structural and functional Plant growth and development are the common re- Plant growth and development are the common physiological processes during growth. Stress (Larcher 1995) is defined as a significant change in environmental conditions relative to optimal condi-tions for growth. It causes changes and responses at all functional levels of a given organism. Quantifying the impact of specific environmental stressors is difficult because: 1) some stress factors affect plants permanently, others only intermittently, 2) the importance of individual stress factors varies at different stages of development, 3) the correlations between changes in environmental stressors and plant growth do not necessarily have a clear cause-and-effect relationship, 4) the growth response to stress may only become apparent after a long period of time, when physiological pro- cesses are altered to the extent that they affect growth rates or developmental processes, 5) the growth responses depend on prior adaptation (pre-conditioning) to changes in the environment, 6) the effects of stress on growth depend on the vitality of the plant, 7) the changes in plant morphology that occur over time interact with the physiological processes that regulate growth. Individual environmental factors can be analysed physiological state. Even very brief and mild stress at this under controlled laboratory conditions, which greatly time will stimulate abnormalities in the physiology of the reduce the variability of results and these are easily plant, drastically affecting growth and development during reproducible. It should be noted that not all environmental this critical period and causing seed and seedling dieback. stressors are harmful to the plant. For example, mild drought The embryo is an essential component of the stress can reduce subsequent damage from transplanting seed, therefore much of the care in handling and storage and gaseous pollutants, affect fruit and wood quality, is devoted to maintaining conditions that will keep the stimulate flowering and induce frost resistance. Storing embryo vital and ready to germinate at sowing or planting. the fruit under controlled conditions, e.g. low temperature The start of embryo growth and its development into a or increased CO 2 concentration, affects their quality and seedling involves most of the life processes that are extends the time period of marketing. important for a plant: water uptake, respiration, conversion Woody plants are most susceptible to dieback of nutrients into soluble forms, synthesis of enzymes during the dormant embryo stage in the seed and during and hormones, metabolism of nitrogen and phosphorus, the development of cotyledons in seedlings. Natural translocation of carbohydrates, hormones, water and regeneration of forests is therefore more dependent mineral nutrients into the meristem, and the consumption on the environmental conditions that are suitable for of nutrients for the development of plant tissues. maintenance of seeds and cotyledons in a suitable 10 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines 2.2.1 Anatomy and Chemical Composition of Woody Plant Seeds The seeds of woody plants vary greatly in size, shape, colour and structure. The smallest ones are barely visible, while the largest ones can reach a few kilograms in weight. Their surfaces can range from smooth to wrinkled. The seeds may have appendages such as wings, fleshy sheaths – arils, thorny vestures, papillas, and hairs. A true seed is a fertilised matured ovule that contains an embryo, a nutrient reserve and a protective seed coat. In practice, the term seed can be used in a broader sense as a functional unit of dissemination. In this sense, it can be a dry fruit with one or more seeds, as well as a true seed. In our seed production practice, we often confuse the fruit with the seed. For example, an acorn is not just a seed but also a fruit, except that the carpel sheath is thin, woody and does not open. The same is true, for example, of beechnut (Figure 1) and elm samaras. The seeds contain different quanti- Carpel ties of nutrients in the form of carbohydra- sheath tes, fats and proteins. Starch is stored in Seed starch granules in the secondary endosperm coat of angiosperms, the megagametophyte of Embryo: gymnosperms and the cotyledons of many plumule, angiosperms. Starch is the most abundant re- hypocotyl, serve carbohydrate, but some gymnosperms radicle Cotyledons store it in small quantities. The most abun- dant reserve sugar is sucrose, while trehalose is found in beechnut, and stachyose and raffi- nose in legume seeds. Fats are found in fat bodies in the form of oils or fats, depending on the ratio of Figure 1: Beech nut in cross-section (photo by Melita Hrenko) saturated to unsaturated fatty acids. At least three-quarters of the proteins in the seed are stored in aleurone grains, organelles 1-20 µm in diameter that are surrounded by a single membrane. In the cotyledons of beech seeds, the reserve proteins originate from a series of divisions of vacuoles in which the reserve proteins are deposited. In addition to protein, protein bodies may also con-tain mineral nutrients and crystals. Some proteins are distributed in the nucleus, mitochondria, proplastids, microsomes and cytosol. Most proteins are metabolically active, but some are not. In addition to protein, seeds contain other nitrogen compounds, amino acids and amides. Other components of seeds are minerals, phosphorus compounds, nucleic acids, alkaloids, organic acids, phytosterols, pigments, phenols, vitamins and plant hormones. 2.2.2 Germination Germination is the initiation of embryo growth, different in shape from the true green leaves, but are capa- which causes the seed or carpel coat to burst and a young ble of photosynthesis. After emergence, not all cells divide as plant to grow through it. Embryo growth requires cell divisi- they did in the embryo, but division is restricted to the meris- on and their growth. When germinating, the radicle develops tematic zones of the apical meristems of the stem and root. into the seed root, the plumule into the shoot, the apical me- Rapid seed germination is usually highly desirable ristem, which produces the stem. With hypogeic germination, because the seed is exposed for a shorter time to infestation subterranean and near-ground germination, the hypocotyl is by insects, fungi, adverse weather conditions and the feeding not extended and the cotyledons remain underground (oak, habits of birds and rodents. However, in temperate zones, it walnut, chestnut). With epigeic, aboveground germination is often better for a species' conservation and dissemination (gymnosperms, beech, hornbeam, ash, most maples, black strategy not to germinate immediately, but to produces locust), the hypocotyl elongates and the cotyledons develop large quantities of seed as a seed bank. The most important above ground into green leaves or needles, which can be very environmental conditions that affect seed germination 11 From Seed to Seedling are water, temperature, light, oxygen, various chemicals and the interactions between these factors. The environmental conditions needed for germination are often more critical than the factors needed for subsequent seedling growth. Heavy-seed plants usually have large nutrient reserves, therefore they can survive post-germination stress more easily than plants with small seed and limited nutrient reserves. Some of the environmental conditions that influence seed germination are discussed in more detail below. Water and drought stress: Non-dormant seeds must take up, or imbibe, a certain amount of water before the physiological processes involved in germination can begin. The absolute amount of water needed is usually small, equal to two to three times the weight of the seed. After germination, water must be available at all times and its required quantity increases with growth and transpiration. Virtually all vital seeds are able to take up sufficient water from the soil at the field water holding capacity of the soil. The effect of soil moisture on seed germination is species-specific. It often depends on the prevailing temperature. Seeds that germinate in the optimum temperature range are less sensitive to water stress. Flooding stress: The start of the biochemical processes necessary for germination depends on the availability of water and oxygen. The soil oxygen content decreases when water fills large pores in the soil. In general, immersing seeds under water for a short time stimulates germination, while long-term immersion in water causes a decline in seed vitality. Underwater seed survival is also species-specific. Temperature: Dormant seed may need a low temperature to break dormancy, after which it needs a much higher temperature to germinate. Non-dormant seeds can germinate at low temperatures, but take much longer to do so. The minimum, optimum and maximum temperatures required for germination are species-specific. They also vary between different seed lots. The range of optimum germination temperatures may vary widely for some species, others have a narrow range, while some can germinate in one temperature regime, others need day/night temperature changes. Light: The species and its origin (tropical species, temperate species, etc.) determine the necessary light, dark or light-dark regime. Most seedlings (of both gymno- and angiosperms), which need light to germinate, germinate fastest when they have a light period of 8 to 12 hours. The wavelength of light is important for some species of ash, birch, elm and some gymnosperms. The wavelength is detected by plants through the phytochrome (a pigment complex that detects mainly red light and, to a lesser extent, blue light, which is better detected by plants through other receptors). As a rule, red light (red, R, 650 nm) stimulates germination, while long-wavelength red light (far red, FR, 730 nm) stops it. The wavelength of the last light flash is important when changing between the two wavelengths. In forest ENVIRONMENTAL FACTORS AFFECTING GERMINATION: Additionally, the litterfall mainly transmits FR light, which • drought stress or the opposite, flooding stress • stands under the tree crown, the ratio of R to FR is small. • water availability temperature can inhibit germination. Germination is influenced by the • light right temperature can germinate even at a lower R:FR ratio. • substrate suitability • physiological state of the seeds – imbibed seeds at the • oxygen availability Oxygen: The early stages of germination are • phytotoxic chemicals allelopathy characterised by intense respiration. Therefore, the seed usually needs higher concentrations of oxygen to germinate than the seedling does to grow. The relatively high oxygen demand originates from the seed coat, which acts as an oxygen diffusion barrier. Oxygen plays an important role in the respiratory chain, but it can also act as an inactivator of some inhibitors of germination. Germination substrate: Germination substrates vary widely in their physical characteristics, temperature and availability of water and mineral nutrients. Mineral soils are generally good substrates because they have a high capacity for infiltration, aeration and close contact with water molecules. Peat and decomposing woody debris are suitable germination substrates, probably because of their water-holding capacity. The suitability of the litterfall depends on the type of plant, the amount and type of litterfall and the prevailing environmental conditions. The litterfall affects the plant community both directly (through impact on seed germination and seedling survival) and indirectly (through effects on the availability of light, water and mineral nutrients). Chemicals: The phytotoxicity of various herbicides, fungicides, insecticides, growth regulators, secondary metabolites, fertilisers and salts can inhibit seed germination or have toxic effects on young seedlings. Phytotoxicity 12 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines depends on the chemical used, its concentration, the plant species, environmental factors and the method of application. Absorption in the soil can cause long-term effects on plants, resulting in abnormal growth and mortality of seedlings. Allelopathy: Seed germination and seedling growth are often suppressed by natural chemicals secreted by other plants. Toxic compounds can be extracted from roots or aboveground parts by gasification, leaching, root exudation and decomposition of plant tissues. They contain various phenylpropanoids (phenols, coumarins, quinones, tannins, etc.), terpenes (resins, saps, essential oils, etc.), alkaloids and organic cyanides. 2.2.3 Germination Physiology Germination includes: 1) imbibition of water in the seed, 2) increased respiration, 3) accelerated enzyme synthesis and degradation, 4) conversion of energy reserves into adenosine triphosphate, 5) altered activity and ratios of plant hormones, 6) decomposition of reserve nutrients and transport of soluble products to the embryo, where cell components are synthesised, 7) accelerated cell division and cellular enlargement, 8) differentiation of cells into tissues and organs. The order of these changes in the seed may vary and may overlap. But the first stage is always water uptake. Some of the changes in the seed are explained in more detail below. Imbibition: The seed must first take up water imbibition and also varies during the three phases of in order to increase the hydration of the protoplasm and imbibition listed above. High energy consumption is also start the series of metabolic processes associated with required when dormancy of the seed is broken. germination. Imbibition also softens the seed coat/carpel Enzyme synthesis and degradation: The enzymes sheath, the seed extends so that it bursts and the seed root are present in dormant seed, but there is a large increase in can grow out of it. Imbibition takes place in three phases: their activity at the start of germination. This is caused by en- 1) rapid imbibition due to physical hydration of zymes changing from an inactive to an active form, new en- the protoplasm, cell walls and colloids occurs in zymes being synthesised after imbibition, and enzymes being dormant, non-dormant, vital and non-vital seeds; degraded and new enzymes synthesised at the same time. 2) this is followed by a congestion phase in imbibition Phosphorus metabolism: FThe phosphorus com- as the water potential of the seed increases (water pounds in seeds are nucleotides, nucleic acids, phosphate potential depends on osmotic potential, turgor esters of sugars and phytin. They are important for energy and matric potential), during which the metabolic conversion and the activation of enzymes in metabolism. activity of the seed increases dramatically; Reserve nutrient metabolism: Germination is 3) the third phase occurs only in germinating seeds, associated with major changes in the metabolic processes first due to an increase in the cell volume of of the nutrient tissues. Cells that originally synthesised the seed root and later due to changes in water insoluble starch, proteins and lipids now hydrolyse them. potential. During seed development, the seed is transported to the Respiration: Respiration involves the oxidative reserve tissues; during germination, it is transported from degradation of organic compounds, mainly sugars, starch, these tissues to the cotyledon meristems. The sources and fatty acids and triglycerides, to produce the energy in the sinks change. form of ATP (adenosine triphosphate) that is required for germination. ATP is used to synthesise new cellular IMBIBITION – WATER ABSORPTION components in the seedling and to synthesise enzymes to The seed is moistened – it takes up water by diffu-break down nutritional substances. The oxygen consumption sion. of dry seeds is very low, but quickly increases sharply with 13 From Seed to Seedling 2.2.4 Seed aging The lifespan of a seed varies greatly, from a few ge instructions for this seed therefore state: days to several centuries. The survival of seeds in the soil 1) for every 1% decrease in the moisture content of the is important from an ecological point of view, in particular seed, the storage time is increased by a factor of 2; for successions and the emergence of weeds. Short-lived 2) for every 5.6 °C lower storage temperature, the sto-seed is seed with a high water content, e.g. in the genera rage time is increased by a factor of 2; Taxus, Populus, Ulmus, Salix, Quercus, Betula and Aesculus. 3) the sum of the storage temperature (in F) and the Depending on their dessication capacity, seeds are divided relative humidity (%) should not be greater than into orthodox (capable of being dried) and recalcitrant (do 100 if the share of temperature is less than half. not survive drying). High-vitality seed tends to germinate quickly, Seed that does not survive drying cannot be dried seedlings grow rapidly in nature. Expressing the vitality of under a relatively high level of humidity without rapidly the seed depends on genetic composition, development, losing its vitality. Even in a perfectly hydrated state, it loses production, storage conditions and the environment during its vitality rapidly (within weeks or months). These include, germination. As the seed ages, its vitality declines: germi- for example, oak acorns, walnuts, hazelnuts, chestnuts, nation and resistance to micro-organisms are reduced, the horse chestnuts. seed root is shorter during germination, the cotyledons fail Seed capable of drying can be dried to a very low to grow outside the seed coat and finally the seed withers. moisture content (normally up to 5%, but possibly up to 2% Similarly, structural, biochemical and genetic changes are of fresh weight). For a given genotype, the loss of vitality of taking place. The latter include changes in DNA, cytoplasm, seed capable of drying, which is stored for a longer period, RNA and chromosomes (e.g. chromosome cleavage). depends on time, humidity and temperature. General stora- 2.2.5 Seed Dormancy Most seeds of woody plants from temperate Causes of dormancy include: climates have some degree of dormancy (resting seeds). ा an immature embryo, This ensures that the seed does not germinate, regardless ा testa impermeability, of favourable external conditions. ा mechanical resistance of testa against embryo Seeds that do not germinate when harvested are growth, characterised by primary dormancy. Those that are able ा metabolic causes in the embryo, to germinate when harvested, but lose this ability later, ा a combination of the aforementioned causes, develop secondary dormancy. This can be triggered by ा secondary dormancy. specific environmental conditions, e.g. very high or low Some of the causes of dormancy and how they temperatures, burial in the ground, anaerobic conditions are intertwined are shown below. and immersion in water. Testa dormancy: Causes can be water and gas Dormancy can be an advantage for the seed, as impermeability of the testa, mechanical barrier of the the longer periods of time at low temperatures needed seed root, retention of inhibitors in the embryo and in- to break dormancy can prevent germination in temperate hibitor production. It is common in legumes and also in climates before spring. The seed of some species survives some pines and apple trees. in the soil for several years, allowing the species to survive Embryo dormancy: Sometimes the embryo is even in the event of late frosts or droughts in previous immature and needs to mature (e.g. Viburnum, Ilex, Ginkgo). years. In places with a dry and wet season, dormancy More often, it is morphologically mature but not capable of prevents germination during the short wet season. If germination inhibitors are present in the testa, they are washed away by rainfall, thus allowing germination to take place in the wet season. At the same time, dormancy is also a nuisance for nursery farmers. It is therefore of both physiological and practical relevance. 14 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines regrowth and germination (apple, oak, chestnut, pear, syca- embryo is morphologically perfect, but must grow to at more maple, some pines, cypresses, larch, fir, juniper). Physi- least twice its size during imbibition before germination ological dormancy develops in two stages: for example, an can take place. However, the growth of the embryo is inhi- ash seed can germinate if it is collected before it desiccates bited by the testa, which hinders access of oxygen. Growth naturally on the tree. The double dormancy is the result of is therefore inhibited until the outer seed coats start to a mechanical barrier of the testa and inhibitors: blackthorn, decay. Even a fully grown embryo is dormant and cannot juniper, yew, holly, Swiss stone pine, etc. In the genus Acer grow from seed until it has been exposed to frost for a few some species exhibit testa dormancy (e.g. sycamore maple), months. Therefore, the first spring germination is inhibited while in others (e.g. Norway maple) embryos exhibit all sta- by the growth of the embryo and by the hindered access of ges in between the two types of dormancy. oxygen. The frost exposure requirement is reached in na- The complexity of dormancy occurring in seeds ture in the second spring and germination can start in the is evident, for example, in the case of ash seeds. The ash second spring after the seed has fallen. Germination may (European and narrowleaf ash) has a dry fruit, the sama- also be slower, delayed by slower testa decomposition and ra, in which the seed is dormant, and germination usually insufficient freezing in the second winter. starts at least one season after the seed has fallen. The SEASON SUMMER WINTER SUMMER WINTER SPRING Process Seed Embryo grows Freezing of Changes in the nutrient reserve GERMINATION production embryo Testa decomposition Onset of dormancy Immature embryo Causes of dormancy Hindered oxygen access Freezing of embryo Diagram 1: Combination of dormancy types in European ash seeds (Kozlowski and Pallardy 1997) Hormones and seed dormancy: In the past, seed tion, but this is mainly due to meristem activity. Auxins dormancy was attributed solely to plant hormones, but are also not associated with dormancy. There are obvio- the mechanisms are more complex. The ABA concentra- usly complex interactions between hormones and other tion (abscisic acid, a stress hormone associated with wa- endogenous factors in the tissues around and inside the ter stress, senescence and growth inhibition) is correlated embryo, as well as environmental factors. with the dormancy rate of apple seeds. The ABA concen- Breaking of dormancy: Dormancy can be broken tration often drops during stratification, but this is not ne- by procedures that affect the metabolism of the embryo, the cessarily related to the breaking of dormancy. Gibberellins, oxygen and water permeability of the testa and/or reduce which have also been linked to dormancy, are probably the mechanical resistance of the testa to embryo growth. Su- mainly involved in metabolism during germination and ccess rate depends on the type or combination of dormancy growth. Cytokinin levels usually increase during germina- types, but for some seeds dormancy cannot be broken. 15 From Seed to Seedling Procedures: ा seed ripening (interplay of light and WHAT INFLUENCES THE BREAKING OF DORMANCY: ा • stratification (low or high temperatures in stratification at low (or alternately low–high) tem-a humid medium or changing temperature temperature), • seed ripening peratures in a humid medium or environment, conditions) ा with chemicals (hormones, peroxide), • chemicals (hormones, peroxide) ा • scarification (mechanical or “acid-softened” scarification (in concentrated sulphuric acid or mechanical testa treatment), testa) ा with heat. 2.2.6 Seed Quality Analyses On representative samples of the seed, the following analyses are carried out: ा purity (% of pure seed, abnormally small seed, mechanically damaged seed, insect-damaged seed, rodent- damaged seed, fungus-infested seed, empty seed, seed of other species, impurities resulting from cleaning of the seed (e.g. wings, parts of flowers, twigs, etc.), impurities of mineral origin (e.g. sand), other impurities), ा mass of 1000 seeds, ा moisture content (%), ा vitality (%, for dormant seed: cut seed test, isolated embryo test, seed X-rays images, colour tests: indigo carmine, tetrazolium – TTC), ा germination test (performance depends on the species; germination is expressed in %, and the germination curve over the time interval and the time taken for 50% germination provide additional information on the physiology and, in particular, the ageing of the seed). The results of a germination test can be expressed in different ways, including germination percentage, germinative energy (refers to the percentage of seeds in the sample that have germinated in a test up to the time when the number of seeds germinating per day reaches its peak), and germi- nation capacity (total number of seeds in the sample that have germinated in a test, plus the number of seeds remaining ungerminated but still sound at the end of the test, in %). Internationally comparable methods are mainly based on the International Seed Testing Organisation (ISTA) method. They are usually carried out in four replications of 100 seeds each. For the purposes of inspection and professional control, the maximum number of seedlings produced per kilogram of seed (depending on the % germination or % vitality and the % purity of the seed) may also be calculated. 2.2.7 Emergence of Seedlings velopment of the seedling, as they store reserve nutrients GERMINATION CAN BE: • The cotyledons are crucial for the growth and de-and minerals, photosynthesise actively and transport the on the earth's surface, and are mainly storage HYPOGEAL: Cotyledons remain underground or substances needed for growth to the apical meristems. organs for nutrient reserves. derground or on the earth's surface, and are mainly sto- grows faster than epicotyl. However, even in species with epigeal germination, the Hypogeal seedlings: • EPIGEAL: In epigeal germination, hypocotyl Cotyledons remain un-rage organs for nutrient reserves (Figure 2). The reserves aboveground cotyledons of some species can are sufficient for development until the first leaves are store significant amounts of reserve nutrients. fully developed. These take over the photosynthetic fun- ction until the true green leaves are fully developed. Leaf of spare carbohydrates. For example, pines accumulate development and the number of leaf shoots per year de- only small amounts, but very quickly start photosynthetic pend on the environmental conditions. activity. After germination, the pine seedling is a sink for Epigeal seedlings: The physiology of epigeal carbohydrates. The synthesis site shifts continuously from seedlings is more species-specific. The above-ground the cotyledons to the first needles and then to the second cotyledons of some species can store significant amounts needles, and reserves are drawn first from the cotyledons 16 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines and then from the first needles for the development of a suitable temperature, primordia of primary needles the second needles. During the cotyledon stage, seedlings immediately develop and grow. are particularly susceptible to environmental stress. The Species without endosperm have cotyledons growth of primary needles depends on the availability of adapted for both nutrient storage and photosynthesis assimilates from the cotyledons. Low temperatures or low (e.g. black locust). Cotyledons of species that have an light intensities during the cotyledon stage can prevent endosperm also transfer nutrients from the endosperm the normal development of primary pine needles. After to the growing tissues. Early seedling development can transferring the seedlings from a cold environment to be divided into three stages (Figure 3): (a) (b) 1 2 3 Figure 2: Schematic representation of (a) subterranean (hypogeal) and (b) aboveground (epigeal) germination (after Kozlowski and Pallardy 1997) (1, 2, 3: emergence phases) 1) in the first phase, from the time the cotyledons emerge from the soil, expand and develop, assimilates are transported from them mainly to the hypocotyl and the root; 2) in the second phase of development, i.e. until leaf development, the assimilates move from the cotyledons mainly to the apical meristem of the stem and the young leaves; 3) in the third phase, as the cotyledons age, the function of supplying assimilates to the roots is transferred to the first true leaf. The development of cotyledons from storage, transfer, photosynthesis and ageing functions requires major changes in enzyme activity. Cotyledons of different species also store, use and take up mineral nutrients differently. In the next phase, they grow to their full-grown size, using up all nutrient reserves for meristematic zones, cell differentiation and chlorophyll synthesis. During this time, the cotyledons undergo positive net photosynthesis. As they age, they begin to yellow and finally fall off. Their lifespan varies depending on the species, and is usually shorter in the case of gymnosperms than in the case of angiosperms. Figure 3: One-year oak sapling (drawing by Eva Margon) 17 From Seed to Seedling 2.3 Tree Nursery Basics The growth and survival of seedlings depend on is required in cold storage facilities. As such conditions their quality, which in turn depends on the quality of the stimulate mould proliferation, they need to be inhibited seed, tree nursery practices and the management of the by low temperatures. Suitable storage temperatures range seedlings during manipulation from the tree nursery to from +3 to -6 °C. Attention should also be paid to the planting in the forest. photoperiod, as there is a practice of storing seedlings Seedling quality is the result of several in constant light. The date of extraction, the start and the physiological and morphological characteristics. Individual total time in cold storage facility are also important. The signs include. dormancy status, water and nutrient ratios, longest the seedlings can be stored is eight months in the morphology, root growth potential, and frost and drought High North. During storage, carbohydrate reserves are used resistance. Individual signs are difficult to evaluate in up, with consumption rates of 0.4 to 0.6 mg per gram per different ways. Seedling size and thickness at the root collar day measured for some conifers. are very useful, but in specific growing conditions, e.g. on Handling seedlings: Transplanting of “bare-root” arid sites, a small seedling with a small transpiration area seedlings is only allowed during the dormant stage of the may perform better than a large seedling. seedlings. The roots should remain moist at all times and Tree nursery practices should be aimed at the air temperature should be low, even in the areas where preserving those physiological processes of the seedling they are buried in the ground and sorted. Frail seedlings that will help it to grow well and survive in the forest. For or those that have suffered insect-damage should be example, the loss of a large number of fine roots during removed. Exposure of the roots can cause growth inhibition transplanting can cause dehydration (desiccation) of and affect seedling survival in the short term. Even after the transplanted seedlings. Successful transplanting planting in the wild, roots are often exposed to water loss and planting in the wild requires an appropriate root to because they grow too slowly to keep up with transpiration aboveground part ratio and rapid root growth in a large soil losses. Transplant shock can take a long time, up to several volume that allows rapid absorption of water and mineral years. nutrients. Nutrient reserves are important because plants Growth potential of seedlings after planting can only regain positive net photosynthesis several weeks in the wild: The growth of the seedlings depends on the after transplanting. Producing quality seedlings requires species and genotype of the plants. Many gymnosperms attention to all phases of tree nursery work: soil and grow much more slowly than angiosperms. The differences bed preparation, planting method and density, fertiliser probably occur because of species-specific photosynthesis, application, irrigation and disease and pest control. In leaf area index, leaf longevity and the distribution of addition, root pruning is sometimes necessary (if this does assimilates in the plant. Growth is also affected by not damage the oblique roots, does not open up areas for competition with other plants, e.g. grasses strongly inhibit pathogens to enter, and minimises the impact on genetic the growth of conifer seedlings. diversity caused by possible root damage to faster- and deeper-rooting seedlings), as well as colonisation with mycorrhizal fungi. LEAFLET: Storage of seedlings The protocol for handling of forest tree seedlin- often requires adequate cold gs is described in a leaflet published under the storage facilities where seedlings can wait in a dormant LIFE project LIFEGENMON (LIFE ENV/SI/000148) (in stage until they are ready to be planted in the wild. They Annex 1). must not dry out during storage, so a humidity of over 85% 18 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines AND SEED PRODUCTION THE PHYSIOLOGY 3 OF FLOWERING IN FOREST TREES 19 The Physiology of Flowering and Seed Production in Forest Trees Studies of flowering and fruit production in forest trees are much more difficult compared to herbaceous plants. Trees are mostly too tall to study the physiology of flowering under controlled conditions, have a long juvenile period before flowering, long reproductive cycles, complex flower bud composition and an unpredictable pattern of flower initiation. 3.1 Flowering Periodicity during their development, which can range from one to Many tree species do not flower every year - the Trees go through a relatively long juvenile period flowering periodicity is both hereditary and more than 40 years in different species (Table 1). dependent on environmental conditions There is a young (juvenile, vegetative) stage and an adult (mature, reproductive) stage, also known as the reproductive stage, when the tree is in “full bloom”. The flowering are particularly large in deciduous trees, but transition to this phase is species-specific and depends smaller in coniferous trees. The formation of cones, fruits on the cultivating conditions. After the transition to the and seeds is a climax phase during long reproductive reproductive phase, the tree retains the ability to flower, cycles that can be influenced by many different factors. and the flowering periodicity (Table 1) varies between and Although the periodicity of flowering is well documented, also within species, depending on the genetic makeup of its causes are not sufficiently explained due to the complex the individual tree. Differences in the periodicity of the interactions of several endogenous and exogenous factors. Table 1: Onset of flowering age and its periodicity in some tree species (adapted from Owens 1991 and USDA 2008**) TREE SPECIES AGE AT THE ONSET OF SEED PRODUCTION PERIOD INTERVALS BETWEEN SEED YEARS (years) (years) Acer platanoides 25–30 1–3/1** Alnus glutinosa 15–20/6–7** 2–3 Betula pendula 15 1–3/2–3** Fagus sylvatica 50–60/60–80** 5–15/3–20** Fraxinus excelsior 25–30/15** 3–5/1–2** Populus nigra** 8–12 1 Populus tremula** 8–10 – Quercus petraea 40–50/40** 2–5/5–7** Quercus robur** 20 2–4 Abies alba** 25–30 2–3 (4–6)* Abies grandis 40–45/20** 3–5/2–3** Larix decidua 25–30/10** 3–5/3–10** Picea abies 30–35/40–60** 3–5/4–13** Pseudotsuga menziesii 30–35 5–7 var. glauca** 20 3–10 var. menziesii** 7–10 (in case of production 20–25) 2–11 * At higher altitudes. ** According to USDA 2008. 20 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines 3.2 Reproductive Cycles Three reproductive cycles are known for most tree species from the temperate climates: A) The most common cycle is two years from the ovule development through a period of winter dormancy, to seed release. Reproductive buds are developed in summer and pollination occurs the following spring. Usually, it takes only a few weeks between pollination and fertilisation. Embryo and seed development is rapid and uninterrupted. Mature seeds can be released as early as late summer in the year of pollination. Seed retention over this time is usually determined by climatic or biotic requirements, which are species-specific and depend on how the seed of that species is disseminated. B) The second reproductive cycle is similar to the first, except that usually it takes a year between pollination and fertilisation. C) The third reproductive cycle is also similar to the first, except that after the embryo and seed development starts it is interrupted in late summer or autumn. The immature seed overwinters and development continues the following spring. 3.3 Initiation and Stimulation of Flowering The initiation of flowering involves the transition environmental factors acting on different plant organs. of an indeterminate vegetative terminal or axillary apical Most measures to improve flowering involve meristem (apex) to a determinate reproductive apical changes in environmental factors: the number of possible meristem, which develops into a flower or flower shoot in combinations is large and their effects are often unreliable or gymnosperms or a cone in conifers. The classical explanation contradictory. Direct applications of plant growth regulators of flowering initiation in herbaceous plants is that flowering in combination with other measures (fertilisation, pruning, is stimulated in different parts of the plant, from where it thinning, etc.) are also used. is transferred within a few hours or days to the apex, where it passes from the vegetative to the floral meristem. Most FLOWERING INITIATION plants blossom depending on environmental factors such Most plants blossom in accordance with factors in their environment, such as photoperiod as photoperiod and temperature. In woody plants, however, and temperature. In woody plants, however, this “simple” classical model does not hold, because this “simple” classical model does not hold, as flowering depends on a series of developmental processes, flowering depends on a whole series of genetically which are sequentially determined by the hormonal and/ conditioned developmental processes. or mineral balance in the substrate, and modified by The morphology and anatomy of the reproductive buds are species-specific, and the timing and location of the flowers are relatively uniform within each species. The environmental factors of particular interest include the following: A) Temperature. High summer temperatures tend to stimulate flowering, which was already described for beech by Linnaeus in 1751. Similar impacts have been described for birch, pine, fir, spruce and Douglas fir. The effects of high summer temperatures, often combined with the application of certain hormones (mainly gibberellins) and drought stress, have, for example, been studied in spruce seedlings cultivated in a greenhouse. B) Light intensity and photoperiod. Most studies show that the effects of light intensity are indirect, depending on crown illumination, terrain gradient, shading and the genetically conditioned distribution of flower buds in the crown. Branches that are more exposed to high levels of light tend to flower more abundantly. Thinning has stimulated flowering in some pines, Douglas-fir and some fruit-producing species. In the case of maple, canopy density influenced the distribution of male and female flowers. In tree species, photoperiod does not have as direct an effect on flowering as in herbaceous species. For three pine species, flowering was not dependent on photoperiod, while in spruce it affected the development and number of cones. In pot experiments, where the effects of light and photoperiod can be separated from those of temperature, it has been found that birch, 21 The Physiology of Flowering and Seed Production in Forest Trees spruce and pine seedlings grown in continuous light or long daylight flower faster than control seedlings (at 10-12 months compared to 4-5 years), and also much faster than trees grown in the wild (15-20 years) (data compiled by Owens 1991). They concluded that the transition to the reproductive phase in these tree species depends more on reaching a certain size than on the number of annual growth cycles. C) Water stress and roots. In pine, spruce, fir, Douglas fir and beech, a positive correlation was found between increased cone/beechnut production and low rainfall. However, roots also play a more direct role in stimulating flowering, not just an indirect one through the uptake of water and nutrients. This role is mainly in the production of plant hormones, cytokinins and gibberellins, which are synthesised in the roots and transported to the aboveground shoots. More direct results on the impacts of drought stress on flowering can only be obtained in pot experiments. They found that a drought stress of -1.4 to -2.0 MPa before dawn has an effect on higher cone production in Douglas fir. Root pruning, or even flooding of the root system, can impact the root activity cycle and hence the flowering. D) Mineral nutrients. In otherwise equal conditions, trees growing on better soils produce more seeds than trees on poorer growing sites. That is why the use of fertilisers is one of the oldest measures to stimulate flowering. In conifers, the main nutrient that impacts flowering is nitrogen. The form of nitrogen (ammoniacal or nitrate) depends on the tree species. In tree species with symbiotic nitrogen fixation capacity (alders, legumes), the use of phosphate fertilisers has been tested with different effects. E) Other stress factors. Any damage to the tree can stimulate flowering, therefore various tree girdling, tying and strangulation techniques are used to accelerate flowering. These procedures are intended to increase carbohydrate concentrations in the crown by inhibiting transport towards the roots. The theoretical starting point is to promote a high C/N ratio, which favours the development of flower buds and inhibits the development of vegetative buds. Opinions on these methods vary. Induction of flowering can also be the result of pruning of branches, grafting, bending, wounding, resin accumulation, defoliation and other biotic influences (pathogens and pests), cold damage and damage to the root system, but there is no simple explanation for the effects of all these stress factors. The lack of positive relationships between en- is important and should precede the development of re- vironmental factors and flowering may be related to en- productive buds. Combinations of other plant hormones, dogenous factors in the tree. A good seed year is usually e.g. auxins, cytokinins, abscisic acid and ethylene, can followed by a year with no or poor seed production. The either stimulate or modify the plant's response to gibbe- growth and development of reproductive organs consume rellins, but on their own they are usually without effect. a lot of metabolites, therefore it may take a year or more Hormonal regulation of flowering and endogenous plant for the tree to regain a condition suitable for the deve- hormone content in buds, shoots and roots at different lopment of reproductive shoots. The appropriate internal stages of development remains one of the most intere- conditions must be matched by external environmental sting problems in basic studies of plant physiology. Howe- factors, which is why the periodicity of flowering and seed ver, after hormone treatment, if appropriately timed and production is relatively infrequent in many tree species. short-lived, the seeds should be of the same quality as Controlling flowering using growth regulators normal seed. serves three purposes: From Section 3.1, it can be summarised that the 1) it prevents early flowering so that most of the onset of fructification is mainly related to the transition energy can be used for vegetative growth and from the vegetative to the reproductive phase, flowering development; period, hormonal regulation and environmental factors. 2) it stimulates flowering of mature trees to increa- Measures to promote flowering can include natural- se fructification; ly adapted thinning of stands, but most other measures 3) it regulates flowering and fructification over can only be implemented in the artificial environment of shorter periods. seed orchards. These include grafted vines, which can help Various gibberellins are mostly used as stimula- plants transfer more quickly into the reproductive phase, tors of flowering in conifers, while these can inhibit flowe- pruning of the crown and roots, girdling, heat or drought ring in angiosperms. The timing of gibberellin application stress, spraying with hormones or injecting hormones or 22 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines hormone inhibitors, and the use of various fertilisers. De- species in the vicinity of the plantation), and the much pending on the species-specific responses of forest trees, better possibilities of manipulation and modification of flowering can be stimulated by a combination of hormonal environmental factors, the development of seed orchards and environmental stress-based measures. However, the has also moved to potted orchards in greenhouses. For effects of symbionts and parasites on flowering are still example, the hybrid larch (Larix x eurolepis) orchard at understudied, not least because of the multi-hormonal the Forest Research Centre in Graupa near Dresden may effects on the growth and development of the plants. be completely covered by netting during pollination, but Due to the high maintenance costs of conventio- otherwise it is exposed to external influences or modified nal seed orchards, the risk of pollination with unselected in terms of temperature, light and water regimes in an un- basic material (naturally occurring trees of the same covered or partially covered greenhouse. Photo 2: Spruce (Picea abies subsp. obovata) cones (photo by Hojka Kraigher) 23 OF FOREST BASICS 4 GENETICS Basics of Forest Genetics 4.1 Mendel’s and Morgan’s Laws Genetics studies heredity in organisms and va- fertilisation, only the dominant trait is expressed, but not riability of genes. Each gene in the DNA of an organism the recessive trait. Homozygotes have identical alleles for has a specific site (locus) on a particular chromosome. a particular trait, while heterozygotes have two different In sexual reproduction recombination of the genes of alleles for the same trait. two parents occurs, resulting in genetically different off- According to the law on independent as-spring. Sexual reproduction is the main source of genetic sortment each pair of alleles is independently distri-diversity (variation) in offspring; the second is mutati- buted (segregated) into gametes. Mendel explained the ons. Adaptation to environmental conditions during the law by observing the inheritance of two independent process of evolution is based on genetic variation in the traits (the colour and shape of the seeds of the pea, the population. Genetic diversity within a population is the pods and the size of the peas). The F2 generation has basis for evolution by natural selection. four possible phenotypes in a 9:3:3:1 ratio. Mendel's laws of inheritance express rules for the random distribution of independent trait characteristics. BIODIVERSITY and phenotype are usually not so simple. In incomple- Adaptation to environmental conditions during the GENETIC DIVERSITY - THE BASIC LEVEL OF However, the relationships between genotype process of evolution is based on genetic variation in te dominance, the heterozygote shows an intermediate the population. Genetic diversity within a population phenotype between the two types of homozygotes. In is the basis for evolution by natural selection. codominance, a heterozygote expresses the trait chara- cteristics of both its alleles. In a population, many genes exist in a large number of alleles. The ability of a single In 1860, Gregor Mendel proved that parents pass gene to affect multiple (apparently independent) phe- on genes to their offspring that retain their identity over notypic traits is called pleiotropy. Epistasis means that a series of generations. He observed hereditary traits that one gene influences the expression of another. Some of showed different characteristics in pea varieties that cou- the characteristics are quantitative and vary continuo-ld undergo true cultivation, i.e. the offspring were all of usly. This suggests that there is a multi-gene inheritan- the same variety after self-fertilisation of the parents. The ce, for the additive effect of two or multiple genes on a traits tested in crossing of two varieties were observed single phenotypic characteristic. Quantitative characte- from the parent generation (P) through the first generati- ristics, which are also influenced by the environment, are on of offspring (F1), then by self-fertilisation of these into multifactorial. the next generation of offspring (F2, etc.): 75% of the F1 ge- The theory of inheritance was further developed neration offspring showed dominant traits, 25% recessive by Thomas H. Morgan, who located genes on chromo-traits, i.e. a ratio of 3:1. Mendel explained the law by the somes. Linked genes are inherited together, not indepen-existence of two alternative forms of the same gene, two dently, because they are located on the same chromo- (or more) alleles, with each offspring inheriting only one some (and often physically adjacent). Each chromosome gene (one form or one allele) from its parents. During me- consists of hundreds of thousands of genes. These genes iosis, two homologous chromosomes of the two parents are linked and are not inherited independently. Some cross and diverge, and the second division produces four genes are linked to sex chromosomes and their inheri- offspring, each carrying one pair of chromosomes and tance does not follow the rules of Mendelian genetics. one series of alleles on each chromosome. The segrega- The phenotypic effects of some sex-linked genes depend tion of alleles for a particular trait from two parents into on whether they originate from the mother or the father. different gametes – the offspring – is explained by the law of segregation. In development, only the dominant allele is expressed, or only when two recessive alleles - linked, sex-linked, chloroplast and WHICH GENES DO NOT FOLLOW MENDEL'S LAWS: are distributed the expression of a recessive trait takes mitochondrial genes place. If two alleles of a certain gene are different after 26 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines Also the genes that are not linked to chromosomes in Where the cytoplasm in the zygote originates from the egg the nucleus do not follow the rules of Mendelian geneti- cell alone, some of the phenotypic traits of the offspring cs: mitochondria and chloroplasts have their own genes. depend exclusively on maternal cytoplasmic genes. 4.2 Population Genetics, Evolution and Speciation For the most part, each population produces A population that is consistent has a gene pool many more offspring than can survive in the environment. described by Hardy-Weinberg equilibrium or equation. These offspring are different and carry different hereditary The simplified form, which describes only two alleles, traits. Between 1840 and 1858 Charles Darwin conceived indicated by p and q, is: the theory of the evolution of species (evolution through modification) based on natural selection. Natural selection p2 + 2pq + q2 = 1 is based on varying survival and reproductive success in a population, leading to the evolution of species adapted The equation allows us to calculate the frequency to different niches in the environment. Environmental of individual alleles in a population's genetic pool if we influences on gene expression can additionally lead to know the frequency of the underlying genotype, and vice (also heritable) epigenetic modifications (Nanson 2004). versa. The Hardy-Weinberg equilibrium is maintained in A population is a limited group of specimens of the population:: the same species. A species is (in a simplified manner) ा if the population is very large; a group of populations whose individual specimens ा if it is isolated from the remaining populations; can reproduce among themselves and produce fertile ा in the absence of inheritance of mutations; offspring (under natural conditions). Each species has ा if there is random sexual reproduction; a geographic range, in which individuals are unevenly ा in the absence of natural selection. distributed, mostly clustered in a few populations. A population may be isolated from others, so that genetic Microevolution is the change in the frequency of material is exchanged only occasionally. Alternatively, a alleles (or genotypes) in a population from one generation population may directly pass into another in a transition to the next. Causes of microevolution include: zone, but even in these populations, individual specimens ा genetic drift – random variations in allele in the centre of the range are more likely to interbreed frequency in the genetic pool (due to random than those at the edge of the populations. Therefore, they sampling of individual specimens), variations are generally more similar to each other in the centre of because of a bottleneck – an extreme reduction their distribution than to individual specimens from other in population size, or variations because of the populations. The entire aggregate of genes in a population impact of forming a new population with a very at a given time is called the genetic pool. It consists of all small number of starting specimens; alleles at all gene loci in all individual specimens of that ा gene flow – a population gains or loses alleles population. Diploid species have each locus represented through the migration of fertile specimens or twice in the genome of an individual, which can be either gametes between different populations; homozygote or heterozygote for that homologous locus. If ा mutations – changes in the DNA of a particular all individual specimens of a population are homozygotes specimen; for the same allele, the last one is fixed in the genetic ा non-random sexual reproduction – individual pool of that population. Usually, two or more alleles of the specimens reproduce more frequently by mixing same gene are represented in the genetic pool at different the genetic material of adjacent specimens than relative frequencies. Allele frequencies are reflected in with others, which leads to inbreeding; another genotype frequency. The frequency of alleles (and, in a form is selective reproduction when individual derivative variant, genotypes) in a population is reflected specimens carefully select a reproductive by the term genetic structure of the population. partner, e.g. one as similar to themselves as possible; 27 Basics of Forest Genetics ा natural selection as a mechanism of adaptive evolution GENETIC DRIFT: different characteristics can act as a stabiliser, – the influence of selection on Random variations in allele frequency in the genetic pool (due to random sampling of species. bottleneck - an extreme reduction in population size, or variations because of the impact of oriented either towards aligning or division of individual specimens), variations because of a Of all the causes of microevolution, natural forming a new population with a very small selection is the only one that leads a population to number of starting specimens. adapt to its environment. Other causes can act positively, negatively or neutrally on adaptations to environmental conditions. Natural selection is made possible by genetic adaptive capacity of the species over its entire distribution diversity within and between populations – variation. area, due to speciation of peripheral races and flexibility in Within a population, it is characterised by polymorphism gene transfer between races. Long-distance gene transfer for a particular characteristic. Measures of genetic has proven to be very important, particularly in maintaining diversity are the proportion (%) of loci (genes) that have the species' resilience to climate change and other large- two or more alleles in a population; the average proportion scale disturbances. of loci, i.e. heterozygotes among individual specimens in a It should be borne in mind that adaptation population; the number of alleles (or allele diversity) per involves the entire biological cycle, vegetative and locus; heterozygosity and nucleotide diversity. generative, of a population, but it is often the reproductive Most species show some geographic diversity phase that limits the population's ability to thrive and adapt representing differences in genetic structure between to changes in the environment (Nanson 2004). The current populations. In a geographically widespread species distribution area is largely the result of several factors, such that thrives in several ecological zones, differences as migration routes from ice-age refugia, where the genetic between populations can arise within a few generations pool of the species may have been diverse, often very because of different selection pressures which, over narrow. Therefore, both ecological and historical sources large areas, can lead to the formation of geographical of variability need to be taken into account when assessing races and within these ecotypes adapted to particular the performance and adaptive capacity of a species in a environments, and within these to populations, which given area. Hence, the ecological conditions in each region in forestry are called provenances. Thus, a provenance of provenance do not reflect all the potential threats to the is the population of forest trees – the forest stand in a thriving of tree populations, and natural forest rejuvenation particular geographically restricted area; such adaptive is not always optimal, nor the most productive, nor the best polymorphism also corresponds to genetic polymorphism adapted to the current conditions in that area. The results (Nanson 2004). Besides natural selection, genetic drift of provenance experiments and simulations of possible can also contribute to polymorphism. A specific form of changes in environmental conditions should also always geographical diversity is clinal variation, in which the be taken into account transition from one ecological zone and geographical race A biological species is a population or group of to another is continuous. Genetic diversity is maintained populations whose individual specimens can interbreed in by diploidy and balanced polymorphism – genes are still nature and produce fertile offspring, but cannot produce exchanged between ecotypes and geographical races, fertile offspring with individuals from other species. The maintaining a single – continuous – genetic pool of a evolution of the species is affected by prezygotic barriers species. Marginal (edge) populations of forest trees thrive such as habitat isolation, isolation due to different under restrictive environmental conditions for a particular behaviour/phenology, temporal, mechanical isolation species, therefore they become rapidly specialised and gamete isolation, reduced hybrid vitality, reduced through natural selection, losing genetic diversity in the hybrid fecundity and hybrid collapse. The concept of a process. In these populations, species can form gradually, biological species is not perfect; there are exceptions. i.e. speciation occurs. This has led to the development of other concepts of Thus, the adaptive polymorphism of a species species: morphological, cognitive, cohesion, ecological, over a large geographical area corresponds to the overall evolutionary, etc. 28 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines The origin of species includes: Conversely, introgression can allow separate ा geographical isolation, leading to allopatric or nearly separate species to merge into one in areas of speciation; introgression where natural hybridisation occurs. Such a ा geographical overlap of species, leading to hybrid species is. e.g.. Abies borisii-regis in the introgression sympatric speciation; zone between Abies alba and Abies cephalonica. The ा genetic changes in populations that can difficulty in identifying oaks and oak acorns arises because include: of natural hybrids between pedunculate and sessile oaks, ◊ interspecies hybridisation, Downy oak, sessile and pedunculate oaks, etc. ◊ agamospermia, ◊ self-pollination. PROVENANCE Where the areas of related species overlap, is a population of forest trees - a forest stand in a NATURAL HYBRIDISATION can occur between them. specific geographically limited area. 29 MATERIAL PRODUCTION FOREST REPRODUCTIVE 5 AND TREE NURSERY TECHNOLOGY Forest Reproductive Material Production and Tree Nursery Technology 5.1 Fructification in Forest Tree Species Onset of fructification: A practical rule emphasises that light-seeded trees, such as birch and alder, start pro-ducing fruits at 10-15 years of age, and heavy-seeded species, such as beech and oak, at 50-60 years of age. The timing of the onset of seed production is also influenced by external factors, so that solitary trees start producing fruits much earlier than those in the forest. Periodicity of fructification: The tree seed production is not the same every year, but is mostly periodic. In mast seed years, which follow each other every few years in certain species, fructification is stronger, with little or no seed production in between. Fructification intensity: For the conservation of forest genetic diversity and the economics of seed husbandry, it is important to assess the intensity of fructification. From the seed harvest practice, based on ocular inspection, the cha-racteristics and ranking of the fructification are derived, as shown in the following table. When assessing the ranks of the fructification intensity, the first and fifth ranks are the most significant, while the remaining ranks can shift continuously from the second to the third and from the third to the fourth. Table 2: Assessment of the fructification intensity Fructifi- Proportion of Suitability for cation Description of rank Characteristics branches with harvesting rank seeds (%) 1 no fructification healthy mature trees do not bear fruit or harvesting is only individual specimens and/or individual 0–10 % not branches bear seeds possible 2 poor fructification poor fructification, mainly of marginal and insufficient for dominant trees, very small quantities, poor 11–40 % harvesting quality 3 good fructification, mainly of dominant and conditionally medium fructification marginal trees, poor fructification of the 41–70 % acceptable for remaining trees, poor quality harvesting 4 Strong fructification very good fructification of most of the fruit- economically producing branches of the marginal and 71–90 % viable for dominant trees, good fructification of the harvesting remaining trees, good quantity and quality 5 Massive fructification virtually all branches in the crown of all recommended trees of the selected species show high 91–100 % for fructification, ideal for obtaining FRM of high harvesting genetic diversity There are also more precise methods that can be used for estimating fructification intensity, by: ा analysing the number of fruits per branch from 10-20 trees in a stand; ा counting the number of visible fruits/seeds/cones per branch/crown in a given time (e.g. 30 seconds); ा collecting the seed in gauges of known dimensions, distributed evenly throughout the stand (at least 5 gauges), calculating the amount of seed collected per hectare (useful for wind-dispersed seed); ा the experimental harvesting method is used to determine fructification, particularly in conifers: a few cones from a few trees are harvested, the cones are cut and the proportion of full and empty seed is determined; ा the average method data on the optimum seed production per hectare for a given species, compared with the experimental seed collection per tree or per specific area in a given year. 32 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines Light-demanding tree species, light-seeded species success of the fertilisation or damage the already fertilised and solitary trees produce fruits more often, while shade-to- flower to such an extent that it does not develop into a fruit. lerant species, heavy-seeded species and trees growing in The external factors that most often cause poor fructificati- stands or shade produce seeds less often. It is important to on despite strong flowering are low temperatures, prolonged collect the seed when fructification is strong and save it for drought during flowering or early fruit development, and infe- years when it is poor, or to grow larger quantities of seedlings. station by plant diseases or insects. Therefore, a more reliable Forecasting flowering and fructification: It is useful estimate is mostly only possible in our species when the fru- to be able to predict the year of full fructification in advance. its are well developed, or 15-30 days before ripening. The first, but very unreliable, prediction can be made as early Time of harvest: The timing of harvest can have a as autumn if we are dealing with a tree species in which the decisive influence on the subsequent germination. It is there- flower buds are different from the leaf buds. A higher number fore necessary to know the state of full ripeness of the seeds of flower buds can indirectly predict a stronger flowering. This for each species and the mean and median maturation time is possible, for example, in the wild cherry, where the flower of the seeds of that species in a particular area. buds are distinctly rounded compared to the leaf buds, but is The fruits can also be important food for forest ani- less certain, for example, in the yew, where the female buds mals, therefore it is useful for seed production practice to co- are quite similar to the leaf buds. llect them as soon as they are sufficiently ripe. For example, Later estimates of fructification intensity based on for wild cherry in June or July, for yew and rowans (Sorbus) in flower abundance may be more reliable. It is important to September or October, and for wild pear in October or No- bear in mind that pollination (even with pollen from incom- vember. patible species) is often sufficient for the initial growth of the Seed variability: Seeds differ with respect to: fruit, while fertilisation must actually take place for durable ा location in the crown or cone (topophysis), fruit growth. Even at this stage, these are still estimates, as ा age of the stand (cyclophysis), the actual fructification is highly dependent on a number ा site characteristics (periphysis). of external factors that can significantly affect the (lack of) 5.2 Collecting, Cleaning and Sorting of Seeds When collecting seed, the first thing to be deter- with seed roots up to 1 cm long. If the fruit is heavily infested mined is the quantity needed, the ripeness, the duration of by insect larvae (mainly in the case of the Balaninus speci-production and the method of harvesting. Immediately after es), the required quality is achieved by immersing the fruit finishing collection in the forest, it is advisable to clean the in water. It is best to sow the harvested acorns immediately seed before transporting it for final processing for sowing in the tree nursery, as prolonged storage is problematic. or storage. Hand-picking of beechnuts is a time-consuming Collecting seeds from the ground: Collecting from process. The triangular shape of the beechnut makes them the ground is the simplest method to produce seeds. This difficult to grip with the fingers, therefore collectors clear is mainly used to collect heavy seeds/fruits, e.g. acorns, be- the ground under the seed trees in stands before they fall echnuts, chestnuts, walnuts, apples, pears. The work can be off. When the fruits fall off, they are swept up, large sieves performed manually, by raking/sweeping or using special are used to separate the beechnuts from other miscellane- vacuum cleaners. It is also possible to track and use seed ous impurities and the clean, full seeds (fruits) are obtained stocks stored by various rodents. by immersing them in water; the problem with immersing Hand-picking of oak acorns has been quite su- them in water is the sand that is left at the bottom together ccessful. Choosing the right time to collect is important be- with the full seeds. Air-dry beechnuts, which still have aro- cause the empty and grubby ones are the first to fall off und 30% moisture, are almost fully cleaned with a windmill. before the healthy ones. The harvesting interval depends on It is stored on the ground in a ventilated area in a layer no the climatic conditions, temperature and humidity, as the more than 10 cm high until shipping. Daily stirring is ne- seeds germinate quickly in good weather (in particular in cessary, otherwise the beechnuts will get mouldy quickly. the case of sessile oak). It is also permissible to collect fruit Immediate sowing is best. 33 Forest Reproductive Material Production and Tree Nursery Technology Tree picking: The seeds on the tree can be collected from the water surface, especially in lowland collected by shaking the tree, climbing, shaking and cutting floodplain forests, using nets and sieves. the branches, or the tree can be felled for the purpose of The effect of seed collection: PAverage daily collecting. quantities of seed when collecting from standing conifer There are various aids for climbing: ladders, foot trees: larch 10-15 kg of cones, pine in selected seed stands 15- rungs or crampon-like climbing irons, tree bicycle (Ger. 50 kg, fir 30-80 kg, spruce 40-100 kg, and from cut trees and Baumvelo), a safety harness, a climbing rope with a pulley individual trees with a low crown 40-60 kg for larch, black system for ascending and descending the rope, appropriate pine 80-100 kg, Scots pine 30-50 kg, fir 80-100 kg and spruce clothing and personal protective equipment, and perhaps 100-150 kg of cones. For deciduous trees, the quantities are a crossbow or a manual swinging mechanism for attaching as follows: pedenculate and sessile oak 30–80 kg, beech 20– the rope to the crown. In Slovenia, ladders are usually 50 kg, sweet chestnut 20–40 kg, walnut 20–40 kg, ash 30–70 used to climb tree trunks without branches, and climbers kg of samaras, sycamore 15–35 kg, black alder 30–80 kg of are secured with a safety harness during collecting. Picking cones, grey alder 10–15 kg of cones, birch 25–50 kg, elm 4–10 from standing trees is one of the most hazardous jobs in kg, hornbeam 15–25 kg, linden 10–25 kg of ripe or 2–5 kg of forestry. Individual climbing equipment manufacturers green fruits and cherry 10–20 kg of stones. also specialise in tree climbing equipment, which is also Some difficulties occurring in seed collection: The promoted in various competitions. biggest problem is obtaining and verifying information on Maple and ash fruits (winged nuts – samaras) are fructification of each species. This leads to a lack of time collected by shaking the tree, climbing, knocking down and or poor coordination in selecting stands for approval and cutting branches with fruit or from felled trees. It is best to collection, particularly in autumn when seed of several collect from standing trees. The pickers spread tarpaulins species is collected. The problems with collecting conifer under the tree, and a person climbs up the tree and knocks seed are the dangers of the work and the relatively few them down from the branches with a stick. On the ground, strong fructification years. Seeds can be stored for longer they separate the leaves from the fruit by hand and further periods, particularly in the case of larch, spruce and pines, clean them using screens. These fruits are ready for sowing. but fir seeds can also be stored for several years; however For storage, they are dried and stored in plastic or jute bags. occasional shortages may occur. The fruits of the wild cherry, which must be Much greater difficulties arise when collecting seed pitted the same day or they will ferment, destroying the of deciduous trees. For species where the seed is collected germinability, are also picked by knocking the branches. from the ground, the main problems are the abundant They are then soaked twice in water to remove empty and understorey, the shrub layer and the stony character of the grubby fruits. As the stone still retains a lot of moisture, the soil. Another issue is the past management practices of seed is dried in a shady and ventilated place for at least the seed stands or their selection, which has mainly been three weeks if it is to be stored, otherwise it is immediately based on the appearance of the tree and the stand, which transported to the tree nursery after soaking in water. is often too dense and the crown can be poorly developed The seeds of conifers (spruce, fir, black pine, larch) and produces few seeds. This is most noticeable in the case are in the cones, which have to be plucked from the branches. of seeds collected by knocking at the branches. In such a Picking from standing trees is one of the most difficult jobs stand, it is virtually impossible to get to the seed. in forestry. Climbers use climbing irons, firefighters' safety Seed collection by cranes and mechanised shaking harnesses and lightweight collapsible aluminium ladders to is not yet an option in Slovenia, due to the high investment aid them in climbing. In the crown, the picker ties a safety costs and the small amount of seed that is collected. harness around the trunk, plucks the cones with his hands However, nets or vacuum cleaners to collect acorns and and stores them in nets or throws them on the ground. His beechnuts from under the trees are being increasingly used. assistant picks them up in sacks and carries them to the truck road, where they are stored until they are transported LEAFLET: The protocol for the collection of seed ma-to the drying plant. Afterwards there may be problems with terial, plant parts and plants from natural regenerati- removing the resin from the skin. on is described in a leaflet published under the LIFE Water surface collection: project LIFEGENMON (LIFE ENV/SI/000148). Alder seeds can be 34 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines Collecting seed when trees are felled requires a seed is not blown out of the drying oven. Larch cones that lot of coordination, from the owner's agreement to cut the are full of resin need to be further rasped after drying, as appropriate number of trees, to the time of felling when otherwise about a third of the seed remains trapped. the seed is ripe, to the presence of collectors at the site Extraction by cracking the fruits: This is used for (taking into consideration occupational safety), and to the walnut, hornbeam and various alien species. willingness of the team to collect the seed immediately after Extraction by removing the fleshy outer part of felling, as it can be completely picked overnight from various the fruits: For sowing, the small fleshy fruits can be dried deciduous trees by wildlife. and used whole. Very fleshy and dehiscent fruits should Seed extraction: Seed extraction can be done be pitted immediately to prevent fermentation and redu- from cones or fruits. It is necessary to facilitate imme- ce germination, for example, the seeds of cherry, apple, diate processing and storage, to reduce spoilage and for rowan, pear, sorb tree, birch, hawthorn, yew, etc. These appropriate manipulation before and after sowing. fruits can be crushed or carefully ground first, but only Extraction by drying: The cones or fruits can be enough to avoid damaging the seed. They are then rinsed air-dried or dried in a drying chamber. Individual conifer vigorously with water. Care must be taken to avoid anae- species can be dried in the sun and at relatively high tem- robic fermentations in the pulp or in the water with the peratures, whereby special covered screens are used in soaked fruit. The water-cleaned fruit is also already pro- which the cones open (with daily turning) and the seed perly prepared. falls through the grille in a relatively short period of 4-5 Cleaning and sorting the seed: This includes all sunny days. After drying, the seed is cleaned, if necessary, the procedures for removing impurities and sizing the se- to remove cones, wings and other miscellaneous impuri- eds. Specific procedures are used for removing the wings, ties. Drying in a fan drying oven is possible at 45-55 °C for sowing and sorting the seed. The seed is usually distri- conifers, but seed from a large number of deciduous trees buted by size using vibrating nets and sieves of different should not be dried at a temperature higher than 20 °C. sizes. Water rinsing and immersion in water are commonly The wind speed must also be regulated to ensure that light used to remove empty seeds. 5.3 Seed Drying The seed is a living organism and any error in its Drying is one of the most demanding processes in manipulation can affect changes in vigour, germination and seed manipulation. For storage, it is advisable to dry the seed dormancy. Each individual extraction and storage process as much as possible, and the degree of drying should be de- has to be studied under laboratory conditions and develo- termined on a species-by-species basis. The drier seeds can be ped under quantitatively completely different conditions in stored at a lower temperature and for a longer period of time. practice. In addition to the species, the physiology of the se- Drying must be started immediately after cleaning eds from different seed lots, different accessions, collected or removal of the carpel sheath, at low temperatures, prefe- in different seed facilities in different years and with diffe- rably between 15 and 20 °C, with air flow, at a low relative hu- rent quality of the crop, also differs. As regards the time and midity (ideally below 10% relative humidity (RH)) in the room, methods of collecting the seed, with particular attention be- in thin layers, up to the specified percentage of moisture. For ing paid to ensuring that the parent stock is not damaged, example, for long-term storage, e.g. for 10 years according to cleaning, the various ways of pre-treating the seed and other the literature, beech seed is optimally stored at 8-9% humi- finishing procedures, particular mention should be made of dity and at -5 to -10 °C. A difference of 1% in humidity may the drying process. result in a disproportionately large change in the germinati- on of the seed after storage, or a 1% increase in humidity may require a storage temperature of about 1 °C higher. SEED The following formula quantifies the relationships is a living organism that emerges from a flower after between seed survival, storage time, temperature and mois- fertilisation and is used to spread through the area. ture content: v = K 2 i – p / 10 K E – C W log 10 m – C H t – C Q t, 35 Forest Reproductive Material Production and Tree Nursery Technology where v is the probable percentage survival of the seed a dry, covered area with air exchange. The layer of acorns after p days of storage at m% seed moisture and t °C, K i is must be no thicker than 15 cm. If it is too thick or there is the seed lot-dependent constant, and K E, CW, CH and CQ are no air exchange, inflammation can occur. Because of high the seed type-dependent survival constants. This equation humidity, the acorn respires intensely when it is collected, shows how the survival of orthodox seed species in air- releasing heat. If it is not drained quickly enough, the dry storage depends on the environmental conditions in temperature in the acorn can rise up to 70°C, leading to which they are stored. inflammation and then loss of vitality. The acorns need to be In practice, the moisture content of the seed can stirred frequently to dissipate heat and dry more efficiently. be tested during drying or weighed to determine whether it Before permanent storage, it is dried to a moisture content has reached the desired dryness (DM%). The initial moisture of around 45%, but the actual moisture content depends content (M%) must be known and is determined by weighing, on the seed lot; the moisture content of the acorns at the drying at 103 °C for 17 + 1 hours and re-weighing (according time of dropping from the trees varies clinally from west to to ISTA 2000 protocols). The control of drying to the desired east, where it is generally a few % lower than in the west, humidity is based on the following formula: and storage is therefore also possible at a lower moisture seed weight (g) at the desired moisture content DM % = content of approximately 40.5% (Žitnik 2003). (100 – initial The process of rehydrating the seed at the end M %) × initial seed weight ( g ) of storage is also important, as it can be mechanically (100 – DM %) damaged by imbibition (water absorption into the seed). Example: 125.3 g of seed with a moisture content of 52.1% Therefore, the conditions prescribe a process in 100% RH, are to be dried to a moisture content of 15%. but not in direct contact with water. Calculation: weight of seed at 15% humidity: ((100 – 52.1)%) However, each process needs to be specifically / ((100 – 15)%) × 125.3 g = 70.61 g developed in the laboratory and subsequently refined to For example, the drying of acorns is very be put into practice for the processing and storage of large problematic. They are dried by temporarily storing them in quantities of seed. 5.4 Seed Storage Seed storage is one of the most problematic areas decades. In recent decades, the increasing share of deciduous in seed production. Seeds can be stored in the short term trees in regeneration through planting has led to a growing from harvesting to sowing, or in the long term for years need to save their seeds. However, this is problematic. In or even decades. Short-term storage is mainly over one the short term, a possible solution is to wait until the next winter. For smaller quantities, autumn sowing or storage sufficient crop to sow or plant, but in the long term, effective in the wild may also be appropriate, as the investment in storage methods need to be developed that are also simple equipment could be far greater than the potential savings. and inexpensive, as the quantity of seed collected and For larger seedlings, storage under controlled conditions used in Slovenia means that we cannot afford complex and is recommended, as this reduces losses, increases viability expensive methods. after storage and eliminates dormancy more successfully. Seeds are divided into two groups according to their However, long-term (multi-year) storage only makes sense ability to be stored for a long time. The first includes orthodox under artificial conditions. It is used to provide seed in years seeds or seeds capable of being dried, which have a significant when there is no fruiting, as most trees produce irregularly reduction in moisture content during the final ripening phase. over a few years. Due to the negative impact of humans on After harvesting, they can be further dried under controlled nature and climate change, it is also becoming increasingly conditions to a moisture content of a few percent and then important to juxtapose and maintain seed banks for use in stored at temperatures well below 0 °C. Under these conditions, seed production and forest gene bank components, e.g. seed the development of pathogenic fungi and the biochemical banks, in which long-term storage plays a key role. activity in the seeds is almost completely stopped, so they can In the past, spruce seed was the main product be stored for years or decades without any significant loss of stored in Slovenia, and it can easily be stored for several vitality. To this group belongs spruce seed. 36 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines The second group consists of recalcitrant seeds only when the cause of dormancy (impermeability of the or seeds that cannot be dried, which do not experience a seed coat, immaturity of the embryo, presence of growth significant decrease in moisture content during ripening. inhibitors, etc.) has been eliminated. As a result, they have a high moisture content after falling, More than 50% of forest tree and shrub species which can be more than 40%. Under artificial conditions, have dormant seeds. In nature, dormancy is eliminated we cannot reduce their moisture content without under the right conditions. The removal under controlled significantly reducing their vitality. Therefore, they cannot conditions is called stratification, and the removal of the be stored at temperatures well below 0 °C because the mechanical dormancy of the testa is called scarification. water in them would freeze and the water crystals would Dormancy can be eliminated in different ways: by collecting damage the tissue. These seeds are therefore stored at a immature seed, which is then immediately stratified temperature of around 0 °C. Under these conditions, the (ash, hornbeam, small-leaved linden), and by stratifying development of pathogenic fungi and biochemical activity mature seed before, during or after storage. In a simplified is only slowed down, so seeds can usually only be stored form, stratification is carried out by sowing the seed for a few months, or until the following spring, without any and, if conditions are favourable, nature will eliminate significant loss of vitality. dormancy. Better efficiency is achieved by stratification Seeds are also divided into dormant or resting under controlled conditions, which is more suitable for seeds and non-dormant or non-resting seeds. Non- larger quantities of seed because of the investment in resting (non- dormant) seeds germinate immediately equipment. There is cold stratification, which takes place under favourable conditions (heat, humidity), while resting at lower temperatures, and warm stratification, which takes (dormant) seeds germinate under favourable conditions place at higher temperatures, both in a moist medium. There are all possible combinations of these seed groups: ा capable of drying (orthodox) + non-resting (non-dormant), Storage ा capable of drying (orthodox) + resting (dormant), complexity ा is increasing non-drying (recalcitrant) + resting (dormant), ा non-drying (recalcitrant) + non-resting (non-dormant). This makes developing optimal seed storage starch in long-term storage of acorns, methods a very complex and difficult task. At the Slove- storage of acorns at low temperatures nian Forestry Institute, we have contributed, among other down to -9°C (Žitnik 2003), and multi-things, to the development of some methods of seed sto- -year storage of spruce seed (several rage and dormancy removal: the importance of phytic acid experiments). in relation to available phosphorus in the soil, sugars and Figure 4: Black poplar: stages of male flower development (drawing by Marina Gabor) 37 Forest Reproductive Material Production and Tree Nursery Technology 5.5 Work Procedures for Seedling Cultivation in Nurseries Cultivation of forest tree seedlings is a multi-year process, with successive stages of work: • soil preparation, • transplanting of perennial seedlings and care thereof, • sowing, • excavating and preparing the seedlings for collection. Soil preparation includes: Sowing includes: Transplanting of perennial seedlings and ा sowing of green ा analysis of seed purity and their care: manure crops, germination, In the next year of the seedling cultivation the ा autumn ploughing, ा dewinging, following is carried out: ा fertilisation, ा soaking in water, ा spring fertilisation with artificial ा soil harrowing or ा disinfection and protection against fertilisers; tillage, rodents and birds, ा weeding is less frequent as the seedlings ा bed layout planning, ा stratification, are dense enough to cover the area; ा row layout planning, ा sowing, covering and rolling the seed, ा water seedlings only as needed during ा pesticide spraying, ा watering and spraying with fungicides, prolonged dry periods; ा watering. ा hoeing, ा regular protection of seedlings with ा dressing with fertiliser, fungicides against fungal diseases. ा preparation for winter or overwintering. Seedlings are transplanted in spring, except for spruce, which takes place mainly in summer. When transplanting, we need to know what the target growth form of the seedlings is. The number of plants per unit area depends on the number of plants per unit area. 1 + 1, 1 + 2, 2 + 2, 2 + 3, 1/2 or 1 + 2 are the symbols that tell us the age of the seedling: one year and two years. This means that the seedling has grown as a seedling for one year and as a transplant for two years, giving a total age of three years. Conifers such as spruce, pine, Douglas fir, etc., are transplanted after two years because the seedlings are tall enough at that age. Fast-growing species, such as larch, should be transplanted after the first year. These species are grown as transplants for two years. During this time, the root ball is enriched and the seedlings are tall enough to be planted in the field. Deciduous trees are transplanted after the first year. For most species, e.g. mountain maple, ash, beech, pear, hazel, rowan etc., 1 + 2 is the best form of training, but for cherry and black alder 1 + 1 is more suitable. Excavating the seedlings and preparing them for collection: When digging up seedlings, we pay particular attention to: ा mechanical or manual; ा the time from digging to planting is as short as possi-ा different forms of growth and root development; ble, which is particularly important during the dry ा grading according to size and quality, with poor ones spring months; being discarded; ा the seedlings are buried as soon as possible after dig- ा pruning seedlings, tying them into bunches; ging and sorting in the ground; ा storage in a humid burial or cold store (bags, +2°C); ा the seedlings are protected from drying out during transport. SFS, in cooperation with SFI, has prepared a Protocol for the manipulation of forest tree seedlings from the nursery to the planting in the forest in cases where the seedlings are provided by the Slovenia Forest Service funded by the budget of the Republic of Slovenia (Annex 1). A good quality seedling is suitable for the particular site in terms of its origin and form, is healthy and vigorous, and may be colonised with suitable root symbionts in special cases. The appropriate size of the seedling may vary depen-ding on the site; the combination of species in planting is a particular issue; the method of planting should be adapted to both soil and vegetation conditions. Greater attention will also need to be paid to the protection and cost-effectiveness of protecting seedlings from wildlife browsing. 38 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines 5.6 Container Seedling Cultivation Due to the ease of handling the seedlings during the seedlings, which allow the roots to be 'air pruned' transport from the nursery to the forest and the possibility and prevent them from twisting, which contributes to the of planting during the growing season, a certain proportion proper development of the root system; this is because of seedlings are grown in container production. Containers root systems often twist in ordinary containers, which, are restrictive in terms of space for root growth. There is less when transplanted into the forest, leads to improper chance of excessive wetting or drying out of the root systems root development and reduced standing capacity of and of attack by pathogens, since production, at least until such trees, as well as to reduced stability of the future the first transplanting of the seedlings, is carried out under stand. Planting in the forest can also lead to a reduction as controlled conditions as possible in glasshouses and in the number of trees that can be transplanted into the greenhouses, which means that production costs are higher forest. than for bare-root seedlings. Therefore, such production For the reasons mentioned above, it is relatively requires: expensive to raise seedlings in containers, so nursery workers ा building suitable facilities, usually greenhouses made of try to maximise the germination of the seed in containers. plastic or glass, with elaborate ventilation, cooling and In most cases, two or three pre-prepared seeds are sown sun protection systems; in each “cell”, which should have as high a germination rate ा the construction of container stands, which should be at as possible, close to 100%, be as uniform in size as possible least 10 cm off the ground; and have a timed germination. Therefore, for the purposes ा the construction of an irrigation or misting system, of container breeding, size grading of seed, various pre- preferably with automatic irrigation metering and treatment procedures and the removal of seed of poorer dosing, and a water demineralisation system; quality from the majority seed are used, all of which lead to ा planning and protecting seedlings against pests and for a reduction in the genetic diversity of the forest reproductive fertilisation; material thus cultivated. ा the development of suitable container substrates that from the roots when transplanting seedlings, provide grading, certain pre-preparation procedures and the extraction of seed of a different quality from the majority, optimum nutrition and are not too favourable for the are able to retain moisture, can be easily removed Containerised seedling production requires seed size all of which lead to a reduction in the genetic diversity of development of pathogens; the forest reproductive material thus produced. ा the development of appropriately sized containers for 5.7 Mycorrhizal Seedling Cultivation Mycorrhiza is the symbiosis between the fungus It is therefore useful, from the point of view of the success and the plant root and acts as the organ for water and nutri- of planting in the forest, to identify which combinations of ent uptake (Frank 1885, e.g. in Kraigher 1996). In ectomycor- fungal species and strains and forest tree species and popu- rhiza, in which the fine roots of tree species (e.g. spruce, fir, lations are most compatible and suitable for particular sites, larch, pine (ectendomycorrhiza can also occur in pine and and to undertake the isolation and rearing of suitable fungi, larch), beech, oak, birch, and to a lesser extent poplar, wil- the multiplication of their mycelia, and the colonisation of low and alder, where other forms of root symbioses have the roots of seedlings of forest tree species. also developed) are completely surrounded by the fungus, The fungi involved in ectomycorrhizal symbiosis the fungus takes over the function of taking up the plant's belong mainly to the ascomycetes and basidiomycetes, and intake of water and nutrients. Seedlings with roots coloni- some can be isolated and multiplied as mycelia or their spores sed by ectomycorrhizal fungi appropriate to the tree species, isolated for colonisation of forest reproductive material. Some provenance and site are more successful when transplanted species of these fungi are also of commercial interest as edible into the forest; mycorrhizae replace the initial fertilisation fungi, e.g. some species of truffles, boletes, chanterelles and and accelerate growth in the first year after transplanting. other edible and culinarily interesting mycorrhizal fungi. 39 Forest Reproductive Material Production and Tree Nursery Technology Colonisation – Mycorrhization of seedlings can take ा It is also possible to use a soil inoculum, i.e. a place in several ways: naturally occurring mixture of mycelium and fungal ा mycorrhizal fungal spores can be used to “coat” spores present in natural soil substrates that have forest tree seeds that are included in special not been treated with fungicides or heat-treated. nursery sowing films; this process is used, for In Slovenian tree nurseries, most bare-root example, in forest nurseries in the Czech Republic seedlings are naturally mycorrhized. For example, the and Slovakia; Omorika Muta tree nursery naturally contains several species ा spores of mycorrhizal fungal species can be of mycorrhizal fungi, among which the predominant species incorporated into the muddy slurry in which the is Thelephora terrestris Ehrh., which is not particularly useful roots of forest tree seedlings are soaked; for forest tree seedlings, but does not harm them either. It ा spores or mycelium of mycorrhizal fungi species would therefore also be appropriate here to take a planned can be added to the soil substrate in the nursery approach to raising seedlings colonised with selected species or in containers; and strains of mycorrhizal fungi. 5.8 Effects of Nursery Practice on Genetic Diversity – from Stand to Seedling, Genetic Diversity Decreases Within the framework of the EUFORGEN programme initial genetic diversity captured in the seed lot at the time (European Forest Genetic Resources Programme), the of its collection in the stand can only be reduced through Working Group on Forest Reproductive Material has prepared the processes of processing, storage, seedling cultivation a Report of the impact of seed and nursery activities on the and planting back into the forest (Diagram 2). genetic diversity of FRM. The guiding principle is that the Production of FRM Seed processing, storage and germination Tree nursery production and seedling cultivation Planting Diagram 2: The genetic diversity of FRM decreases from stand to planting back into the forest. Forest seed and tree nursery practices that reduce genetic diversity, an excerpt from the EUFORGEN Working Group Report (Gömöry et al., 2021): ा Seed Production: ◊ collection (selection of non-related trees), timing (physiologically mature - immature seed) and method of col- lection (from the whole crown, accessible from the ground, side of the crown, etc.). • Seed processing and germination processes: ा extraction, thermotherapy/chemotherapy/encapsulation, drying, size selection; ा storage, stratification/scarification, germination procedures (in germinators, in the bed); ा germination rate (elimination of later-germinating genotypes). • Exclusion/selection/standardisation of seed and/or seedlings: ा seed unsuitable for seed producers (species purity in the EC Directive); ा for tree nursery operators (standardisation required by the EC Directive only for Mediterranean oak species and poplar cuttings). 40 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines ा Transplanting and pruning to optimise the ratio between the shoot and root system: ◊ uniformity of the root system – seedlings of species with deep primary (oblique) roots are more damaged and affected. ा – Fertilisation and protection against diseases and pests: ◊ hereditary resistance of individual provenances and clones to diseases and pests; ◊ experiments with different N fertilisation regimes – high and optimal N concentrations with differences between families, low concentrations with no differences (several pine species); ◊ differences in growth completion and frost resistance – linked to fertilisation regimes. ा Mycorrhization: ◊ the use of mycorrhizal beech and oak seedlings in Germany – as a replacement fertiliser (Kottke et al. 1987); ◊ dependent on the fertilisation regime, the use of pesticides and the naturally occurring species and strains of fungi in the nursery; ◊ coexistence efficiency, depending on the fungal species and strain and the species and provenance of the forest tree (Gianinazzi-Pearson et al. 1984, Hazard et al. 2017), ◊ use in forest and non-forest plantations (forest by-products; Grebenc et al., forthcoming). ा Storage of seedlings: ◊ the period, duration and storage regime; ◊ In Turkish semi-desert conditions, the presence of heart roots and induced drought stress prior to harvesting increase post-planting survival – impact on increased genetic diversity after successful planting, ◊ growth completion and frost tolerance are hereditary – inappropriate timing of excavation can reduce genetic diversity. ा Vegetative reproduction: ◊ cuttings, somatic embryogenesis (SE), micropropagation. ा Containerised seedlings: ◊ extreme standardisation of seeds and seedlings; ◊ the possibility of planting in different seasons and conditions; ◊ the cost of production – the speed and price of planting; ◊ experience 2017/2018 at Postojna OU: mice. ा Manipulation and monitoring of stand quality after planting (traceability): ◊ a protocol for manipulating seedlings from nursery to forest; ◊ planting methods and timing (conifer-conifer, container seedlings, etc.); ◊ protection of seedlings (game, rodents, use of chemical products, etc.); ◊ planting planning, ◊ monitoring the success of planting; ◊ monitoring the effects of management on the quality of future stands. Figure 5: Black poplar: stages of female flower development (drawing by Marina Gabor) 41 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines REPRODUCTIVE FOREST 6 MATERIAL ACT 43 Forest Reproductive Material Act The European Directive on the marketing of A PREREQUISITE FOR FOREST STABILITY forest reproductive material within the European Union, adopted in December 1999 (EC/105/1999), is a common is regeneration with site-adapted forest reproductive material (for natural rejuvenation or guideline that prescribes the necessary professional reforestation by planting and sowing). supervision and allows a free market. Each Member State shall, on the basis of the minimum criteria set out in this Directive, determine in its national legislation and Regeneration is one of the most crucial phases regulations the responsible operators and procedures in forest development. It is where the hereditary patterns for professional supervision, the demarcation of of the future forest are formed, required for the stability regions of provenance and the suitability of the use of future stands. The prerequisite for forest stability of forest reproductive material therein, and the means is regeneration with site-adapted forest reproductive of maintaining stability and biodiversity in its forests. material (for natural rejuvenation or reforestation by These procedures may include rules on seed reserves, planting and sowing). The need for forest reproductive seed storage, seed banks and the wider forest gene material to be adapted to the site is reflected in the bank in each country. In this chapter we summarise demarcation of regions of provenance and the rules on the essential articles, terminology and procedures of the use of forest reproductive material in them, which the Forest Reproductive Material Act, the requirements are being adapted to the necessary measures for the for registration (approval) of basic material (forest seed genetic conservation of forests in a time of rapid climate facilities) and the procedures for certification of FRM. change. FOREST REPRODUCTIVE MATERIAL ACT (ZGRM 2002) EUROPEAN DIRECTIVE ON THE MARKETING OF includes: FOREST REPRODUCTIVE MATERIAL • the requirement to conserve forest genetic is a common instruction that prescribes resources (FGR) in forests and plantations professional supervision and facilitates free trade outside forests; within the European Union. The basic requirements • the characteristics of traditional and are aligned with the OECD Forest Seed and Plant sustainable multifunctional forest management Scheme. as prescribed by the Forest Act (ZOG 1993). 44 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines 6.1 Interpretation of Terminology According to the Forest Reproductive Material Act (2002) Terminology is mostly based on the European Directive on the marketing of forest reproductive material (EC/105/1999). Reproductive material includes: 1) Seed material: seeds, cones, fruits and co-products intended for the production of planting material. 2) Plant parts: cuttings, cutting material, explants or embryos for micropropagation, buds, layers, roots, substrates, grafts and any parts of plants intended for the production of planting material. 3) Planting material: plants grown from seed (seedlings), plant parts and plants from natural regeneration (plants from natural rejuvenation). Forest seed facilities is the basic material for the production of reproductive material, which may be a group of seed trees, a stand, a seed orchard, parents of family, a clone or a mixture of clones: ा A group of seed trees are trees within a given area from which seed is collected. ा A stand is a spatially restricted population of trees with a uniform composition. ा A seed orchard is a plantation of selected clones or families which is isolated or managed in such a way as to prevent or reduce pollination from external genetic sources and in which the management method allows frequent and abundant cropping and easy seed production. ा The parents of family are trees intended to produce offspring by controlled or free pollination, with one known parent as the mother tree and the pollen of another parent (sibling family) or group of parents (half-sibling family). ा Clones are a group of individuals (ramets) derived originally from a single individual (ortet) by vegetative propagation, for example by cuttings, micropropagation, grafts, layers or divisions; ा A clonal mixture is a mixture of identified clones in defined proportions. Reproductive material categories: 1) “Source identified” Reproductive material of known origin derived from basic material which may be either a seed source or stand located within a single region of provenance and which meets the prescribed minimum requirements. 2) “Selected” Reproductive material derived from basic material which shall be a stand located within a single region of provenance, which has been phenotypically selected at the population level and which meets the defined requirements. 3) “Qualified” Reproductive material derived from basic material which shall be seed orchards, parents of families, clones or clonal mixtures, the components of which have been phenotypically selected at the individual level, and which meets the requirements prepared based on this Act. Testing need not necessarily have been undertaken or completed. 4) “Tested” Reproductive material derived from basic material which shall consist of stands, seed orchards, parents of families, clones or clonal mixtures. The superiority of the reproductive material must have been demonstrated by comparative testing or an estimate of the superiority of the reproductive material calculated from the genetic evaluation of the components of the basic material. Table 3: The categories in which each type of reproductive material can be marketed, depending on the basic material from which it was produced. CATEGORY OF FOREST REPRODUCTIVE MATERIAL Type of Basic Material Source Identified Selected Qualified Tested Seed Source X Stand X X X Seed Orchard X X Parents of Family(-Ies) X X Clone X X Clonal Mixture X X 45 Forest Reproductive Material Act Reproductive material that is marketed must be The seed storage is a store of seed material intended accompanied by a document. If this is coloured, then yellow for use in planting and sowing in the event of a shortage of indicates source identified, green indicates selected, pink indi- reproductive material in forests and constitutes a compulsory cates qualified and blue indicates tested FRM. reserve of reproductive material for the needs of sustainable Autochthonous stand or seed source: An autochtho- forest management in the Republic of Slovenia. nous stand or seed source is one which normally has been The Slovenian Forest Genebank is a controlled or continuously regenerated by natural regeneration. The stand or cultivated population of forest woody plants managed for the seed source may be regenerated artificially from reproductive conservation of species and their gene pools. It consists of fo- material collected in the same stand or seed source or auto- rest seed facilities, including forest gene reserves and genetic chthonous stands or seed sources within close proximity. monitoring stands, special specimens or populations of forest Indigenous stand or seed source: An indigenous trees, living archives of forest tree species, test plantations, a stand or seed source is an autochthonous stand or seed source seed bank and other biological materials. or is a stand or seed source raised artificially from seed, the A seed bank is a long-term collection of representa-origin of which is situated in the same region of provenance. tive samples of seed material from a seed storage and other Origin: For an autochthonous stand or seed source, sources. the origin is the place in which the trees are growing. For a no- Export is any exit of a consignment of reproductive nautochthonous stand or seed source, the origin is the place material from the customs territory of the European Union (va- from which the seed or plants were originally introduced. The lid for the territory of the Republic of Slovenia) and includes origin of a stand or seed source may be unknown. re-export and temporary export. A provenance is a locally defined site of any stand of Import is any introduction of a consignment of re-forest trees. productive material into the customs territory of the European For a species or sub-species, the region of provenan- Union (valid for the territory of the Republic of Slovenia), irre- ce is the area or group of areas subject to sufficiently uniform spective of the purpose of the introduction, with the exception ecological conditions in which stands or seed sources showing of introduction for transit. similar phenotypic or genetic characters are found, taking into Transit is any movement of a consignment of repro-account altitudinal boundaries where appropriate. ductive material through the customs territory of the European Production means any stage in the extraction and fi- Union (valid for the territory of the Republic of Slovenia). nishing of seed material into seed and the raising of planting The master certificate of origin is the document cer-material from seed or plant parts (such as: collecting, harvesting tifying the origin of the reproductive material. and other extraction of seed material, plant parts, direct-use The accompanying document is the document that shoots and buds from the starting material, drying, dehulling, accompanies the reproductive material when it is marketed. cleaning, calibrating, storing, packing, labelling, preparing for A plant passport is a document that accompanies planting, determining quality elements, etc.). plants or parts of plants whenever they move within the EU, in A lot is a well-defined and physically limited quantity accordance with the provisions of plant protection legislation. of reproductive material produced in a given year and in a given A comparative test is a statistically designed experi-forest seed facilities (basic material). ment in which reproductive material is evaluated against one or Marketing means selling or delivering to other per- more predetermined standards. sons, including delivery under a contract for services, displaying A standard is a stand, plantation or plant that shows with a view to sale, offering for sale. better characteristics than the average in a given area and is esta- A supplier (professional operator) is any legal or na- blished as a benchmark before the comparative test is started. tural person carrying out a production activity with a view to marketing or importing reproductive material. A PROVENANCE is a locally defined site of any stand of forest trees. The production of seed and “pullings” (naturally rege-A REGION OF PROVENANCE for a species or subspecies is an area or group of areas nerated saplings collected in approved forest seed facilities for with similar ecological conditions in which stands or further nursing in nurseries for planting in forests) in forests is groups of seedlings have similar phenotypic or genetic a function of the production of other forest goods under the characteristics, taking into account elevational zones. forest regulations. 46 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines 6.2 Minimum Requirements for the Production of “Source Identified” Forest Reproductive Material The basic material must be a group of seed tre- roved according to a prescribed procedure, during which reproductive material production in the category “source identified” in Slovenia include stricter es or a stand growing in one region of provenance, app- Requirements for seed facilities for forest the proposed stand is evaluated against criteria 1 to 7 of criteria than those laid down in European Directive Directive EC/105/1999 and the ZGRM (see Section 6.4 for EC/105/1999. explanations). The minimum number of trees from which FRM is harvested is 25 for most tree species, and optimally at le- The following information must be known and ast 50 trees growing at least one and preferably two crown provided: heights apart. For minority species, the minimum number ा region of provenance; of trees is 10, and preferably more than 25, spaced at an ा a location plotted on a map; appropriate distance from each other to ensure that they ा altitude/elevational range; are not natural ramets of the same clone. ा the bedrock and possibly the plant community; Phenotypic criteria are taken into account in the ा the regional unit, possibly the local unit, the fo-selection: rest management unit, the cadastral municipali- ा for conifers, general adaptation to site conditions, ty, the forest department and its section; straight growth and appropriate crown shape; ा origin, which may be indigenous, non-indigeno- ा for deciduous trees, in addition, twisted growth us, natural or unknown. must be minimised; forking, if present, is only al- In the case of non-native or non-natural origin, lowed in the crown. the origin of the basic material, if known, must be given. 6.3 Minimum Requirements for “Selected” Seed Stands The stand is assessed according to the specific purpose for which the reproductive material will be used. In this respect, the requirements of points 1 to 10 shall be reasonably taken into account. The purpose of the use shall be indicated in the Register of Forest Seed Facilities of the Republic of Slovenia. 1) Origin: It must be established, on the basis of hi- 4) Age and development phase: The stands must be storical sources or by other appropriate means, of such an age and stage of development that the whether the selected stand is of indigenous/na- selection criteria can be assessed unambiguously. tive, allochthonous/non-indigenous or unknown 5) Uniformity: Trees in stands should show a normal origin. For allochthonous/non-native, the origin degree of variability in morphological characters. If must be given, if known. necessary, poorer quality trees shall be removed. 2) Isolation: Stands shall be sufficiently distant 6) Adaptability: The stands must show adequate from stands of inferior quality of the same tree adaptation to the ecological conditions in their species or variety which may form hybrids with area of origin. This is expressed by the ability of the tree species in the selected stand. This is the population to thrive permanently on its site, i.e. particularly important when the surrounding ve- to rejuvenate successfully, either generatively or getation is of non-native or unknown origin. vegetatively; generative rejuvenation means that 3) Population size: The stands must be composed flowering, fruiting (respecting periodicity), natural of one or more groups of trees with an appropri- rejuvenation (seed germination) and survival of ate distribution and in sufficient numbers to al- the young saplings are observed in the stand. For low adequate cross-pollination. The number and non-native species, the potential invasiveness of density of trees in a stand should be sufficient to the species or the forest tree population being as- eliminate the undesirable effects of inbreeding. sessed is recorded under this criterion. 47 Forest Reproductive Material Act 7) Health condition and immunity: Trees in stands conditions and that are managed in a similar way. must generally be free from pests and diseases and 9) Wood quality: The quality of the wood must be must be resistant to adverse climatic and soil con- taken into account when selecting stands; under ditions (pollution is excluded) on their site. On par- certain conditions, it can also become a decisive ticularly unfavourable sites, specific characteristics criterion. can also be identified regarding the resilience of the 10) Growth form: Trees in stands should show good forest tree population to such conditions, e.g. lead morphological signs of growth, in particular trunk contamination of the soil, etc. elongation and symmetry, adequate canopy growth, 8) Volumetric increment: In general, the volumetric in- thinness of branches and their adequate natural di- crement of a stand should be greater than the ave- e-back. In addition, the proportion of forking trees rage of the other stands growing in similar ecological and trees with spirally twisted timber should be low. 6.4 Forest Seed Facilities for the Production of the “Qualified” and “Tested” Categories of FRM FRM of the category “qualified” is produced from organisation of the work and the planting design, i.e. the seed or clonal orchards. For the purposes of designing such minimum number of clones and replications of the same a plantation, a register of plus trees should be prepared clone in the plantation, the spatial distribution, the tending for each tree species, corresponding to the phenotypic and thinning system, and the flowering and pollination characteristics determined at the tree level. In Slovenia, the control system. The plan with all the necessary components criteria for the approval of plus trees for wild cherry and is then approved by the Institute and an expert opinion is black poplar have been elaborated, but the register is still issued as the basis for the plantation design. Once planted relatively deficient, as the formal requirements for approval and before FRM production begins, this seed facility must be of each tree are the same as for the approval of the whole approved, verifying the adequacy of the planting, the number stand. In addition, agreements must be reached for the of clones and the replication of clones in the plantation. At acquisition of grafts or cuttings from the individual trees the same time, the maximum number of seeds or planting (ortets) for the production of planting material for the design stock allowed from each orchards shall also be fixed. of a seed or clonal plantation (number of clones – ramets per FRM of the category “tested” may originate from each plus tree – ortet). in situ (selected) or ex situ (qualified) seed facilities that For the design of seed and clonal orchards, it is meet predefined standards or have been tested in progeny important to determine the distance from stands of the same tests. This category of forest seed facility has not yet been species that could affect the mixing of genetic material, the registered in Slovenia. 6.5 Production of Forest Reproductive Material The sequence of operations necessary to initiate and control the production of forest reproductive material is shown below. 1) Recording forests with a production function for forest reproductive material is carried out by the Slovenian Forest Service (hereinafter referred to as the Service). the Institute and the responsible personnel from 2) Only a forest seed facility approved by a decree the Service. may be used for production of forest reproductive 3) The procedure for the approval of a forest seed material intended for marketing. The procedure facility shall be initiated on the basis of an for the approval of a forest seed facility is condu- application from the owner or other formally cted by the Slovenian Forestry Institute (hereinaf- acknowledged forest user forests owned by the ter referred to as the Institute) under an admini- Republic of Slovenia is submitted by the Slovenian strative procedure based on an inspection by a State Forests company (SiDG). The forest owner or committee formed by the authorized experts from user may authorise the Service to represent him in 48 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines the procedure for approval of a forest seed facility production of FRM in the 'qualified' and 'tested' in his forest. categories shall be inspected by the Institute at 4) The Institute decides by a written Decree on the the time of flowering (seed orchard) or before the approval of the forest seed facility. The decree start of production. specifies the type of approved forest seed facility 6) For the purpose of approving forest seed facilities and the category of reproductive material to intended for the production of reproductive be produced therein, and shall prescribe the material in the categories ‘source identified' and conditions for the production of seed material and 'selected', the regions of provenance shall be plants from natural regeneration in the forest seed established. The Regulation on the Delineation of facility. Regions of Provenance, of which the Provenance Regions Map is a component, also contains more detailed recommendations for the use of the production of reproductive material in the reproductive material in the individual provenance For approved forest seed facilities to be used for categories “selected”, “quantified” and “tested”, regions and at elevational zones. which are a mandatory content of forest required under the forest regulations, the Service management and detailed silvicultural plans. the institute shall draw up tending guidelines 7) For forest seed facilities, in addition to the data also keeps records on the status of these stands and groups of trees, and on flowering and 5) For approved forest seed facilities to be used for fructification, and reports on these regularly to the production of reproductive material in the the Institute. The Institute keeps records of the categories “selected”, “quantified” and “tested”, the quantity, type and quality of forest reproductive institute shall, upon approval, write guidelines for material produced. tending which are a mandatory content of forest 8) The Institute shall establish and manage a register management and silvicultural plans. The Service of forest seed facilities. Entry in the register shall plans the management of forest seed facilities and be ex officio on the basis of the approval decrees advises their owners on the function of production issued. of seed and pullings. An approved forest seed facility intended for the production of selected The Institute shall establish and manage a register reproductive material shall be inspected by the of forest seed facilities (basic material). Service at least once a year; seed facilities for the Figure 6: Black poplar: female flowers and female catkins with seed capsules (long white silky hairs giving a fluffy and cotton-like appearance) (drawing by Marina Gabor) 49 Forest Reproductive Material Act 6.6 Master Certificate for Forest Reproductive Material If the reproductive material is produced in a forest for inclusion in the forest genebank (extracted DNA for the seed facility under the provisions of the ZGRM, a certificate of DNA library or extracted seed for the seed bank). origin is issued. On the basis of the confirmation of the Slovenia Suppliers must notify the Service (for in situ seed Forest Service and the report on the extraction of seed by facilities) or the Institute (for ex situ seed facilities) in good the professional operator, the Institute shall issue a master time, but at the latest one week before production is due to certificate of origin to the seed dealer – professional ope-start at the forest seed facility, in order to secure the issue rator no later than one week after the end of production in of a master certificate of provenance. They submit an appli- the forest seed production facility or one week after receipt cation for FRM production to the responsible SFS local unit, of the record of the seed extraction from the FRM producer, which also includes the consent of the owner of the seed provided that the conditions laid down in the guidelines wri- facility for production of the FRM. tten at the time of approval of the facility have been met and During production, the Service monitors the pro- that the origin clearly corresponds to that recorded in the SFS gress of the production in the forest seed facility and issues confirmation. a confirmation of FRM production. Before the end of pro- In order to ensure uniform records, the owner shall duction at the forest seed facility, the prescribed quantity notify the time and quantity of the production of reprodu- of plant material (alive twig with three buds, seeds, fruits or ctive material for his own needs to the Service no later than cones) from each tree from which FRM has been obtained one week before the start of production. shall be sent to the Institute (by the Service or the professio- nal Operator), as well as the confirmation with the quantities MASTER CERTIFICATE FOR FOREST REPRODUCTIVE of collected seeds or pullings (e.g. cones or fruit obtained MATERIAL sample of the extracted seed, together with a copy of the forest seed facility under the provisions of the Forest Reproductive Material Act, a certificate of and a record on the extracted seeds) and a representative If the reproductive material is produced in a confirmation to the institute for analysis and inclusion in the origin is issued. active part of the Slovenian Forest Genebank, which is used for FRM certification, for any possible future inspection and 6.7 Forest Reproductive Material Lot At all stages of production, the reproductive material must be separated by lots, each identified by the following information: 1) certificate of origin number and country code; (indigenous, natural, known (indication of origin), 2) botanical name of a species or hybrid of species; unknown); 3) category; 9) the year of seed production; 4) purpose; 10) the age and type of planting material (seedlings, 5) type of forest seed facility; plants for natural regeneration (pullings), 6) the registration number of the forest seed facility cuttings); in the register of forest seed facilities; 11) genetic modification. 7) the provenance region – for source identified and selected forest reproductive material, and for the other categories where appropriate; 8) the origin of the reproductive material Figure 7: Black pine: stages of needle development (drawing by Klara Jager) 50 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines 6.8 Reserves of Forest Reproductive Material basis of contracts on the purchase, storage and ongoing and category of seeds and other reproductive store reproductive material in this way, the Service stores it material that would endanger the stability of In order to prevent a shortage of a certain species renewal of reproductive material. If it is not possible to forests, the environment, genetic resources and itself in its own or rented warehouses. Slovenia, SEED STORAGE shall be organised within ductive material from the Seed Storage under a concession or the framework of the public forestry service. biodiversity in the territory of the Republic of The supplier or the person entrusted with the repro- storage contract may not pass on or otherwise dispose of the material without the authorisation of the Service. In order to prevent a shortage of a certain species The use of the reproductive material reserves and category of seeds and other reproductive material that is decided by the Service through the Forest Investment would endanger the stability of forests, the environment, Programme. It specifies the type of reproductive material, genetic resources and biodiversity in the territory of the the purpose and manner of use, the time limit and manner Republic of Slovenia, Seed Storage shall be organised of replacement of the used reserves, if any, and, at the within the framework of the public forestry service. time of sale, the price of the reproductive material and the Samples of seed material stored in the Seed portion of it to be used to finance the reserves. Storage are also obligatorily included in the seed bank, According to the Service's programme, planting which is an integral part of the Slovenian Forest Genebank, material grown from the seed reserves is intended for use managed by the Slovenian Forestry Institute. in forest planting in the territory of the Republic of Slovenia. The establishment and use of stocks of reprodu- Funds for the creation and maintenance of the ctive material are carried out by the Service, but individual Seed Storage and for the purchase and storage of the tasks may also be carried out by concessionaires. compulsory reserve of reproductive material are provided The Institute provides guidance for the develop- in the budget of the Republic of Slovenia according to the ment of the Seed Storage and the Slovenian Forest Gene- forest investment programme drawn up by the Service. bank and carries out expert tasks regarding quality control EU funding from the Rural Development Programme can and provenance of the seed material. also be used to help with the rehabilitation of large-scale The minimum quantity and type of reproductive disturbances. material to be stored in the seed bank shall be planned by the Service in the forest investment programme and is Samples of seed material stored in the Seed determined by the Minister by regulation. Storage are also obligatorily included in the SEED The Service stores the compulsory reserve of BANK, which is an integral part of the SLOVENIAN FOREST GENEBANK, managed by the Slovenian reproductive material not covered by the concession as an Forestry Institute. increased commercial stock with major suppliers on the Figure 8: Black pine: stages of needle development (drawing by Klara Jager) 51 Forest Reproductive Material Act 6.9 Comparison of the Competences of National Legislation and EU requirements The EC Directive on the marketing of forest reproductive material (EC/105/1999) establishes a basic level of pro-fessional control and exchange of information on the production and marketing of forest reproductive material, which each EU Member State can build upon with its own national legislation. The European Commission's Directive requests: demarcate regions of provenance; ा control of forest reproductive material of forest ा which provenances of forest trees it will designa- tree species listed in the Directive and used for te for use in forests/provenance regions in its forestry purposes, territory; ा marketing only the prescribed categories of fo- ा whether it will prescribe ways to maintain the rest re-productive material, stability and biodiversity of forests in its territory ा traceability of forest reproductive material at all by making it compulsory to purchase the plan- stages of production and marketing, ned quantities of forest reproductive material ा a clear map delineation of the provenance regi- from seed facilities in its territory in order to set ons and their description, up a seed storage; ा the possibility to freely transfer and market fo- ा whether it will also include in its legislation in rest reproductive material throughout the EU. this area additional content that has nothing to It depends on the EU country: do with the Directive, e.g. content relevant to the ा how it will design the system of supervision and system for the conservation of forest genetic re- authorise organisations for professional supervision; sources, the content and functioning of the Slo- ा which additional forest tree species it will inclu- venian Forest Genebank, forest gene reserves, etc de in its national lists; The demarcation between the European and na- ा whether it will set stricter criteria for the granting tional legislation is shown schematically in Figure 5. Howe- of a decision for approval of forest seed facilities ver, Slovenian legislation is also based on the requirement intended for the production of reproductive ma- to conserve forest genetic resources (under Article 2 of the terial in the category “source identified”; Forest Reproductive Material Act), from which the by-laws ा how it will design the delimitation system and also derive. Diagram 3: Delimitation of the European and national content of legislation on the marketing of forest reproductive material (FRM) and the conservation of forest genetic resources EUROPEAN REQUIREMENTS: Transparent owner-to-customer traceability of FRM and a free market in the EU Designation of authorised bodies National legislation Reserve requirement (organisations) rules Seed facility selection procedure Rules on regions of provenance and the issue of the certificate and use of FRM therein 52 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines Diagram 4: The Slovenian Forest Reproductive Material Act is also based on the requirement to conserve forest genetic resources, which is set out in detail in sub-legislative acts. FOREST REPRODUCTIVE MATERIAL ACT Existing practice The EU Directive and the MARKET Regions of provenance Register of seed facilities FRM certification 6.10 Meaning, Competences and Sequence of the Rules Reforestation is one of the most crucial phases in the lifetime of a forest and one of the most important acti-vities affecting its development. The basic condition for stability is regeneration with site-adapted forest reproductive material (for natural rejuvenation or for regeneration by planting and sowing). The timing of regeneration depends on silvicultural planning or a series of silvicultural measures. This is followed by quality regeneration by planting/sowing, which primarily depends on the physiology and technology of storage, cultivation and planting of forest reproductive ma-terial. The complex of professional guidance is directly linked to and dependent on professional supervision of all phases of the work, i.e. supervision of the origin and quality of forest reproductive material and, in the case of forest nurseries, health checks. The expert guidance and supervision process includes: 1) reforestation with site-adapted forest reproductive material, i.e. a regulation delimiting provenance regions and recommendations for the use of FRM in them, 2) timing, which for seed and seedlings means in particular the timing of the final cutting and reforestation of stands, and the planning of the appropriate quantity and quality of planting material, including measures in the seed facilities, which are part of the forest genebank, and the management of the seed storage, 3) transparent professional control of forest reproductive material based on nationally proven procedures and institutions, including inspection and international cooperation on the transfer of seed and seedlings across national borders. Figure 9: Black pine: stages of male flower development (drawing by Klara Jager) 53 REGULATORY PROVISIONS 7 CONCERNING FOREST REPRODUCTIVE MATERIAL Regulatory Provisions Concerning Forest Reproductive Material Tree populations adapt to local environmental con- nous stands is the most appropriate for planting or sowing. ditions through natural selection. Selection influences the This also prevents the introduction of alien genetic material adaptation of populations to local conditions at the genetic which, through cross-pollination, could affect indigenous ge- level, and the outward expression of differently expressed netic material and weaken the natural stability of stands. To genetic composition is reflected in the phenotype. Therefore, meet this requirement, we define regions of provenance that the genetic variability between individual populations of trees are geographically distinct and within which populations of a must be taken into consideration when planting or sowing. To given tree species have a similar genetic makeup. It is there- maintain the natural stability of stands, the use of reproductive fore recommended that forest reproductive material collected material with a genetic makeup similar to that of autochtho- in a particular region of provenance be used only in that area. 7.1 Demarcation of Regions of Provenance The best methods for delimitation of regions of pulations. Regions of provenances can be subsequently up- provenances are provenance trials and DNA analyses, as the- dated and verified based on the results of provenance trials se methods can determine differences in genetic makeup and DNA analyses. The boundaries of the regions of prove- between individual populations of trees. However, metho- nance are also generally aligned with the administrative bou- ds of provenance trials are time-consuming and logistically ndaries, which are preferably marked in the field, to facilitate challenging, and DNA analyses are not yet available for all the identification of the regions of provenance in the field, to species and populations within the territory of the Republic meet the practical needs of the growers in the individual re- of Slovenia. The current and provisional solution is to select gional SFS units in their planning, and to facilitate the super- regions of provenances based on natural factors (ground vision of planting or sowing. The whole of Slovenia is divided rock, soil, topography, vegetation, local climate, etc.) that are into altitudinal zones, which, even more than the regions of assumed to impact the genetic diversity of individual tree po- provenance, define the suitability for utilisation of FRM. 7.1.1 Seed Districts (1951-1986) The first division of Slovenia into seven regions of collected seed would be used for sowing in the field and in provenances, named seed districts, 1 was made- by Maks Wra- tree nurseries. A system of a large number of forest nurseri- ber in 1950. He stressed the biological basis of forest seed es has been designed, distributed across geographical areas and tree nursery production and the need for a systematic, and altitudinal vegetation zones. The selection of seed stan- guided and supervised seed service, in particular the pro- ds was based on phytocoenological (phytosociological), bio- venance and selection of forest seeds. On the basis of geo- logical and ecological, genetic, systematic, technological and graphical, geological-petrographical, climatic and vegetation economic criteria. The division, based on vegetation units, zones defined on the basis of phytocoenology, Slovenia was represents the basic vegetation division of Slovenia and thus divided into seven forest seed districts: the Triglav, Kam- also reflects climatic impacts well. In the following decade, niško-Savinjska, Pohorsko-Kozjaška, Podravsko-Pomurska until 1961, Miran Brinar also set out detailed principles and (sub-Pannonian), Posavsko-Dolenjska, Postojnsko-Kočevska methods for the selection of seed stands and drew up a de- (mountain forest karst) and Karst Region seed districts. With tailed map for the demarcation of seed districts (Figure 4), the cooperation of the Expert Group for Seed Production and and later the seed-production districts were further subdivi- Forest Nursery Activities, the Forest Management Authority ded into altitudinal zones. and the Slovenian Forestry Institute, a large number of fo- The division of Slovenia into seed-production dis- rest seed stands intended for the permanent production of tricts was also given legal effect in the Seeds and Seedlings quality seed were selected in the field, and seed areas were Act (1973): “Forest seeds and seedlings may be used only wit- identified where quality seed of native forest species was hin the altitudinal zones and seed-growing districts where collected. At the same time, it was also foreseen where the the forest seeds were produced.” 1 The terms seed and seed-growing district were used. For practical purposes of translating Slovenian legislation into English language, the current proposals use the related term regions of provenance, which is more clear. 56 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines Figure 10: Map of forest seed-growing districts of Slovenia from 1971 (Register, 1971) 7.1.2 Seed Units (1986-2002) At the time of the first revision of seed stands in per tree species or group of tree species. The division into 1982-1985, Marjanca Pavle introduced the division of Slo- seed-production units was applied in practice until 2002, venia into seed-production units for the purposes of seed and a second revision of the seed stands was performed production. A seed-production unit is a group of similar on this basis, which was also led by Pavle until 1997. Unfor- forest communities on the same bedrock (carbonate, si- tunately, the distribution of the seed-production units was liceous) and in the same altitudinal zones (0-399 m, 400- inadequate for the mapping of the regions of provenance 699 m, 700-999 m, > 1000 m). Reproductive material col- and difficult due to the high level of fragmentation in the lected within a particular seed-production unit may only field, therefore the demarcation of the regions of prove- be used within that unit. Seed-production units are not nances had to be redefined after the adoption of the ZGRM geographically coherent units, but are fragmented across in 2002, when the seed-production legislation had to be Slovenia. They were determined for each harvesting or brought into line with European requirements. planting and sowing site separately. They were designated 7.1.3 Demarcation of Slovenia into Regions of Provenances (2002-) In accordance with Directive EC/105/1999, the the field by the boundaries between.the regional units – territory of Slovenia was delineated into regions of the forest management regions and the administrative provenance (Kutnar et al. 2002). These are delineated to boundaries – the cadastral municipalities, allowing a continue in the direction outlined by Wraber in 1950 and detailed overview of the production and recommended improved by Pavle in 1987. The improvement is based on the use of forest reproductive material in the individual forest new phytogeographical division of Slovenia as proposed management regions and forest management units Article by Mitja Zupančič and Peter Žagar in 1995. For individual 7 of the Rules on determining regions of provenance (2003) species, they are also delineated on population genetic includes the following guidelines for use: studies. A similar division based on ecologically related areas is adopted in some other European countries, e.g. Germany (BML 1999). The Rules on determining regions of provenance The basis for the demarcation are the broad include recommendations for the use of FRM. ecological zones, which are demarcated in more detail in 57 Regulatory Provisions Concerning Forest Reproductive Material » (1) To guide the utilisation of FRM, the following suitability (2) If the most suitable or very suitable FRM is unavailable scale is used: in the seed facilities of a specific region of provenance 1. Most suitable: FRM from a seed facility within and altitude zone, and it is not available in the seed the same subregion of provenance and altitude vault, FRM for suitable or less suitable utilisation may zone; be stored or utilised, up to a maximum of one year. 2. Very suitable: FRM from a seed facility within the (3) If FRM for less suitable utilisation is unavailable for same region of provenance and altitude zone; more than 10 years, FRM for exceptionally suitable 3. Suitable: FRM from a seed facility in the adjacent utilisation may also be stored or utilised, up to a region of provenance and the same altitude maximum of one year. zone; (4) Regardless of the provisions of the preceding 4. Less suitable: FRM from the same region of paragraphs, for the purpose of preserving forest provenance and altitude zone, but from different genetic resources, only the use of FRM from the Šavrin seed facilities; sub-region of provenance shall be permitted.” 5. Exceptionally suitable: FRM from other regions of This article was subsequently amended several provenance and the adjacent altitude zone in the times, so that, following the latest harmonisation, the same regions of provenance and altitude zone. utilisation of FRM from certain regions of provenance from neighbouring countries (Austria, Croatia and Hungary) in certain areas and altitudes in Slovenia is also permitted, subject to the prior expert opinion of the Institute. Regions of provenance (ecoregions and subregions) 31 11 Julian Alps 12 Western Karawanks - Kamnik Alps 32 13 Savinja Alps - Eastern Karawanks 31 Goričko 20 32 Mura Plains Pre-Pannonian 13 20 Pohorje Pohorje 33 35 Obsotelje Hills Alpine 11 34 36 Krško-Bizeljsko Hills 43 37 White Carniola 24 Haloze - Dravinja Hills 12 33 Slovenske gorice Hills - Ptuj Field 41 Škofja Loka Hills - Sava River Valley 35 42 Posavje Hills Pre-Alpine 42 43 Savinja-Šaleško Region 41 51 Dry Carniola - S Zasavje Hills 52 Mirna-Radulja Hills 51 54 Gorjanci Mountain Range 52 53 Bohor Mountain 61 Trnovo Forest Plateau 51 Pre-Dinaric 71 36 62 Inner Carniola - Snežnik Mountain Range 62 63 Kočevje-Ribnica Mountain Range Dinaric 71 Gorizia Hills - Vipava Valley 54 73 Brkini Hills 63 Sub-72 Karst - Vrem Hills 72 74 Šavrin Hills Mediterranean 37 forest 73 Pre-Pannonian 74 region Figure 11: Demarcation of regions of provenance in Slovenia 58 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines 7.2 List of Species to Which the Forest Reproductive Material Act (ZGRM) Applies Directive EC/105/1999 lists 48 species and artificial From the principle of conservation of forest genetic hybrids subject to the guidelines of the Directive, which resources, it is in the interest of the State to regulate all forest must be transposed into national law; it is permissible tree species used for planting in forests and permanent to supplement the rules set by this Directive, which may forest plantations, including windbreaks, park forests in be stricter than those listed, but for each exception an urban areas, etc. For this reason, Slovenia has already informed application must be made to the EC for a waiver included some additional species in the first species list, in of the specific requirement. The latter was used by Slovenia 2010 it added all indigenous tree species to the list, and since in 2005, when the EC granted Slovenia and Denmark a 2019 efforts have been underway to include a larger number decision to remove individual alien and economically non- of shrub species that are important for the stability of forest viable species from the national list of species subject to ecosystems and whose funding for use would allow them to the ZGRM. be included in the list of species subject to the ZGRM. Table 4: List of species subject to the ZGRM; for clarity purposes, authors of the latin names have been omitted. Abies alba Larix kaempferi Prunus avium Acer campestre Phillyrea latifolia Abies cephalonica Larix x eurolepis Pseudotsuga menziesii Acer monspenssulanum Pinus mugo Abies grandis Larix sibirica Pyrus pyraster Acer obtusatum Pistacia terebinthus Abies pinsapo Malus sylvestris Quercus cerris Acer tataricum Populus alba Acer platanoides Picea abies Quercus ilex Alnus viridis Populus nigra Acer pseudoplatanus Picea sitchensis Quercus petraea Carpinus orientalis Populus tremula Alnus glutinosa Pinus brutia Quercus pubescens Celtis australis Prunus mahaleb Alnus incana Pinus canariensis Quercus robur Cercis siliquastrum Prunus padus Betula pendula Pinus cembra Quercus rubra Ficus carica Pyrus amygdaliformis Betula pubescens Pinus contorta Quercus suber Fraxinus ornus Quercus crenata Carpinus betulus Pinus halepensis Robinia pseudoacacia Ilex aquifolium Salix x spp. Castanea sativa Pinus leucodermis Sorbus aria Laburnum alpinum Taxus baccata Cedrus atlantica Pinus nigra Sorbus aucuparia Laburnum alschingeri Ulmus laevis Cedrus libani Pinus pinaster Sorbus domestica Laburnum anagyroides Ulmus minor Fagus sylvatica Pinus pinea Sorbus torminalis Laurus nobilis Quercus crenata Fraxinus angustifolia Pinus radiata Tilia cordata Mespilus germanica Juglans regia Pinus sylvestris Tilia platyphyllos Olea europaea Larix decidua Populus x spp. Ulmus glabra Ostrya carpinifolia Species deleted by EC Order 2005 Species on the national list, most of them added by SI Order 2010 Alien tree species in the current Order Species prescribed in the European Directive 59 Regulatory Provisions Concerning Forest Reproductive Material 7.3 Procedures for the Approval of Forest Seed Facilities According to the Forest Reproductive Material the following documentation: descriptions of the stands and Act (2002, including subsequent amendments and supple- mapping material: an overview location map at a scale of ments), forest seed facilities (FSF) can include: 1:25,000 and a TP at a scale of 1:5000, with the boundaries ा seed trees or stands of forest trees for the produ- and numbers of the land plots, divisions and sections plo- ction of forest reproductive material of the “source tted, as well as the information on the owner/-s of the land identified” category; plots where the seed facility is located. ा stands of forest trees for the production of forest The commission inspection, led by SFI, shall be reproductive material of the “selected” category; attended by authorised representatives of SFI, SFS and the ा tree orchards for the production of the forest re- owner or the owner's or manager's representative; if ne- productive material of the “qualified” category; cessary, other experts, dendrologists, phytopathologists, ा stands or plantations of trees for the production and seed or nursery personnel shall also be invited to par- of forest reproductive material of the “tested” ca- ticipate in the field inspection. During the field inspection, tegory. the stand is assessed according to the requirements on Approval of FSF for the “source identified” and “se- the information document, the stand/facility information, lected” categories is initiated on the basis of an application the access route to the facility, and the guidelines agreed received from the owner of the seed facility; in the case of a between the SFI, the SFS and the owner/manager or repre- demonstrated need to obtain forest reproductive material, sentative for the tending of the stand and the collection of SFS registers presumably suitable seed facilities and initi- FRM are entered in the assessment sheet; these guidelines ates the process of obtaining an application for approval form a mandatory part of the unit's forest management plan from the owner – communication with the owner depends (FMP). On the basis of the quantification of the criteria in the mainly on the competent district foresters or other locally assessment sheet, at the end of the evaluation of the site or regionally responsible SFS experts. After the date of the and in comparison with the quality of other stands of the visit has been coordinated between the owner, SFS repre- same species in the wider area, a decision is made by the sentatives and those responsible for conducting the com- committee as to whether the proposed site is approved and mission inspection with SFI, the SFS representative prepares in which category (“source identified” or “selected”). 7.4 FRM Certification Procedures Confirmation of the SFS for in situ production of accept it, provide the supplier with a detailed presentation FRM: At least seven days prior to the intended production, of the approved forest seed facility and the requirements the registered supplier must notify the local SFS unit (or, in for the production of FRM in the stand or on a map, and the case of production in a seed plantation, the Institute) shall specify the process of the SFS expert supervision for of their intention to do so by submitting an application for obtaining the SFS confirmation for the in situ production an FRM master certificate (Annex 6); the application shall of FRM in FSF. If the production of the FRM was not carried guarantee the consent of the owner or manager of the FSF out under the supervision of the SFS, the person in char- or his/her legal representative to the production of FRM on ge of the SFS regional unit shall, upon completion of the the property in question. NB: Since the main cost of FRM production, write on the SFS confirmation “Not under the production is the collection, processing and storage of the supervision of the SFS”. FRM, the arrangement is usually verbal, often based on a The amount of FRM produced is signed daily on the commodity exchange agreement, e.g. delivery of a few fruit SFS form by the person in charge in the field, usually the district tree seedlings, etc. forester, and the total amount is certified by the regional head If the production of FRM is in accordance with the of silviculture. During the production process, a sample (plant medium-term reforestation needs of the territory of Slove- tissue – twig with three dormant shoots or seed/fruits/cones) in nia, the application is eligible for exemption of the payment the prescribed quantities (in accordance with the Rules on Cer- of administrative fees. The SFS shall review the application, tificates) shall be taken from each tree from which the FRM has 60 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines been produced, by the SFS expert officer (district officer or head FRM produced in the stand and the quantity of FRM processed. of silviculture) or by the supplier producing the FRM in the FSF, Providing all information is adequate, and the directi- from each of the trees from which the FRM has been produced ves for collection of FRM in FSF have been taken into account, (in accordance with the Rules on Certificates), stored in a paper the SFI shall issue a Master Certificate of Origin (Annex 7) no bag and sent immediately to the SFI, together with a copy of the later than 14 days after receipt of the dossier. If requested by SFS certificate, for the purpose of checks on the origin at the the supplier or the SFS, the SFI may also produce a seed quality time of issue of the master certificate or subsequent identifica- analysis and then a quality report (Annex 8). The quality analysis tion of the FRM. The supplier shall also deliver to the SFI a re- can also be carried out by the supplier themselves according to presentative sample of the entire batch (lot) of processed seed, an internationally recognised methodology (ISTA). together with a processing record showing the initial quantity of 7.5 Overview of the Series of Procedures for Approval of FSF and FRM Certification 1) APPROVAL OF SEED facilities under the General Administrative Procedure Act (ZUP) 1. Stranka The customer submits an application on Agriculture, Forestry and Food (MKGP) from the offi- the prescribed form to the SFI. cial records, which must be received within 15 days. Fee: application – 50 points (tariff code 1), decision 4. In the categories “source identified” and “selected”, – 200 points (tariff code 3). The client is: the SFI obtains an information document from ा the owner; the SFS. The cost of compiling the information do- ा a supplier to whom the owner has assigned the cument is borne by the SFS – Public Forest Service production of reproductive material for a fixed (PFS). or indefinite period of time, by means of a no- 5. SFI prepares: tarised written declaration; ा tending guidelines: for stands (for the category ा any other person authorised to represent the “selected”); owner in the approval procedure. ा guidelines for production: seed tree groups, 2. The application shall be accompanied by: stands; ा a notarised declaration of renunciation of the 6. SFI issues the approval decision with a written production of reproductive material; decree. Time limits for issuing a decision: ा power of representation of the owner; ा shortened declaratory proceedings: one month, ा qualified: management plan; ा specific declaratory proceedings: two months. ा tested: test results; 7. The SFI enters the facility in the register. ा genetically modified: authorisation for release 8. The costs incurred in the approval process shall be into the environment. charged to the client by the SFI. The SFI shall decide on 3. If the application is deficient, the SFI has 3 days to the costs in a decision, whereby in practice all costs are request additional information from the Ministry of covered by PFS. 2) OVERVIEW OF SEED FACILITIES 3) DELETION OF SEED FACILITIES (ZUP) 1. SFS inspects facilities of “source identified” and “se- 1. Ex officio, if the facility does not comply with the lected” once a year. Costs: PFS. requirements. Fee: decision – 200 points (tariff code 2. The SFI reviews the “qualified” and “tested” catego- 3). ries once a year. Costs: PFS. 2. At the request of the facility owner. 3. At the request of the owner, SFS/SFI inspects the The costs of the deletion procedure are borne by facility. Costs: owner. the owner and decided by the SFI in a decision. Fee: application – 50 points (tariff code 1), decision – 200 points (tariff code 3). 61 Regulatory Provisions Concerning Forest Reproductive Material 4) PRODUCTION OF REPRODUCTIVE MATERIAL (ZUP) 1. Seven days before the start of the production of reproductive material (in situ), the supplier shall submit to the SFS a written application from the supplier for obtaining a master certificate from the Institute. For seed and clonal orchards, SFI is included instead of SFS. Fee: application – 50 points (tariff code 1), certificate – 50 points (tariff code 6), certificate – 30 points (tariff code 6). 2. If the supplier is not the owner, the application must be accompanied by: ा a notarised declaration of renunciation of the production of reproductive material; ा a written declaration of a one-off renunciation of the reproductive material; ा power of representation of the owner. 3. During production, the SFS (SFI) periodically monitors the harvesting: ा an inspection report shall be drawn up; ा a sample of the reproductive material, which is forwarded to SFI, is taken ा the supplier enters the quantity of reproductive material harvested on a daily basis on the application form 4. SFS issues the application as a confirmation to the supplier and sends a copy of the confirmation to SFI. The costs incurred in the procedure are charged to the supplier by the SFS by decision (price list established by the Ministry of Agriculture and Rural Development). 5. The SFI issues the master certificate no later than seven days after the completion of the production or com- pletion of the processing. Depending on what the supplier has requested in the application, the SFI issues a Master Certificate: ा as soon as they have received the certificate and the sample from the SFS after the certificate has been issued, a sample of the processed seed is taken for the genebank; ा after processing at the seed establishment, where SFI checks the quantity and takes a sample of the pro- cessed seed for the genebank. 6. The SFI charges the costs incurred in the procedure to the supplier by decision (price list to be established by the Ministry of Agriculture and Rural Development). If all the procedures are for the approval of seed facilities and the certification of FRM for the purposes of me- dium-term planning of the need for FRM in Slovenia in accordance with the plans of the SFS, the procedures are exempt from fees, and the costs of the approval are part of the tasks of the public forestry service. Figure 12: European ash: leaf-out phases (vegetative bud burst phenological phases) (drawing by Metka Kladnik) 62 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines FACILITIES FOREST 8 SEED 63 Forest Seed Facilities Forest seed facilities are approved by the Slovenian Forestry Institute under a modified administrative pro-cedure on the basis of an application from the owner/s and/or operator/s of the facility and a commission inspection to assess whether the facility meets the regulatory criteria. It is assigned an identification number by the SFI, the first number of which indicates the region of provenance (1 to 7, or 0 if the region of provenance covers the entire territory of Slovenia), and is entered in the Register of Forest Seed facilities published in the Official Gazette of the Republic of Slo-venia in January each year and added to the European FOREMATIS list. In 2019, a total of 380 seed facilities were approved in Slovenia, of which 104 for the production of FRM “not for use in forestry”. Among the others, the majority are in the “selected” category for FRM production, fewer are in the “source identified” category, only one seed orchard is approved for FRM “qualified”, and there is no registered seed facility in Slovenia for the “tested” category. It should be noted that the purpose for which the FRM is produced in a particular seed facility may be indicated by: 1 – “for multifunctional forestry”; 2 – “not for use in forestry” or “of limited wood The purpose for which the FRM is produced may be lity is for use in forestry, but is primarily in- 2 – “not for use in forestry”; A footnote indicates if it is a forest genetic reserve production significance” – such a seed faci- 1 – “for multifunctional forestry”; tended for the conservation of forest genetic or a genetic monitoring plot. resources. 8.1 Seed trees or stands for the “source identified” FRM category Due to the need to conserve forest genetic re- up to 40%. Such a seed facility may be a stand or a gro- sources and the scarcity of FRM, an increasing number of up of seed trees; for autochthonous tree species it shall forest seed facilities have been approved in Slovenia for normally be of natural origin, except that for spruce and the production of FRM in the “source identified” category. Austrian (black) pine it may be non-autochthonous or of However, these FSF must also meet the criteria for appro- unknown origin; for seed facilities that are not of natural val as written for FSF of the “selected” category, except that origin, a note shall be added to the effect that the stand criteria 8, 9 and 10 are not taken into account for approval originates from FRM of local origin or from a local tree (see 6.4); the proportion of trees with major defects can be nursery. 8.2 "Selected" Seed Stands Seed material for reforestation by planting in Slo- The practical benefits of selected seed stands venia is mainly produced from selected seed stands. They were already identified by Wraber (1951): represent the best part of the populations of a tree species in 1. ZReliable provenance and good quality forest terms of the characteristics that are important for the future seed are guaranteed. development and yield of that tree species in a harvested “ 2. It is possible to effectively control the collection forest. The objective of seed stand management is tailored and distribution of seed, which must be rational to the role of seed production and includes the production in terms of use within and outside the seed di- of quality seed with excellent genetic composition, while at strict. the same time meeting other forest management objectives. 3. All the racial characteristics of forest trees con- Seed from our seed stands is categorised as “selected” in tained in certain forest stands are preserved and the European Forest Reproductive Material Categorisation strengthened by the correct grouping of the seed Scheme. The provenance, climatic conditions in the area and districts. a series of other data are known, and the starting material 4. The demarcation of forest seed districts and the (the seed stand) is selected on the basis of the phenotypic isolation of seed stands is the scientific and pra- characteristics of the whole population of trees in the stand. ctical basis for the selection of forest seed, i.e. 64 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines for the intensification of good growth characte- 8. Accurate information on the provenance and se- ristics and for the cultivation of as pure a species lection of forest seeds will greatly increase their as possible, with an established hereditary basis value when sold abroad.” or a high vital force. Sustainable, multifunctional and co-natural fo- 5. The grouping of the seed districts determines rest management requires strict adherence to the origin of the appropriate horizontal and vertical distri- the seed and continuous selection of seed material when bution of the forest nurseries and allows for an resowing and replanting. The seedlings, which are formed efficient utilisation of forest seedlings. through reforestation by planting or sowing, must have the 6. Seed districts provide a realistic basis for forest genetic makeup to be able to meet all the forest manage- seed planning, both in terms of absolute quanti- ment objectives of the future forest. The type of measures ty and the quantitative relationship between tree in the selected seed stands is therefore forest tree cultiva- species. tion. It aims to improve the genetic makeup of future fo- 7. Adherence to the previous criteria will increase rest tree populations in line with the intended objectives, the quality of forest seeds and forest seedlings while maintaining a broad genetic diversity that will provi- and, of course, the quality and quantity of forest de the population and the species with security in the face production. of unpredictable changes in the environment. 8.2.1 Description and Tending of Selected Seed Stands The description and analysis of the condition form phenotype of all individuals is the basis for planning mea- the basis for planning care measures in seed stands. A de- sures to improve the genetic composition of the stand or for tailed analysis of the forest stand condition is already carri- cultivating a particular forest tree species. The objectives of ed out in the process of selecting seed stands of individual cultivation of a particular tree species are defined by the traits tree species. Situation analysis involves evaluation and as- that we want to improve in that species, while the cultivation sessment of: programmes define the methods, procedures and selection ा the need for selected seed stands of a given speci- criteria that will be used to achieve genetic improvement. Me- es in the region of provenance and altitudinal zone asures in seed stands are an important part of a species' cul- under consideration; tivation programme. They do not destroy the genetic structure ा information on the site and stand; of populations, but ensure a continuous process of improve- ा the stand based on the phenotypic characteristics ment. For this reason, it is considered that seed stand mana- of all specimens; gement is a major component of the sustainable breeding ा stands of the tree species concerned in the area of forest tree populations and an integral part of “cultivation adjacent to the seed stand. without cultivation” (El Kassaby and Lstibůrek 2009). The basic information includes information on the Morphological and phenological trait characteristi- site, forest community, stand size, timber stock, increment, cs, which are the criteria for selection in oaks, may be more number of trees of the tree species under consideration, or less genetically conditioned. The environment and silvi- stand structure, stand composition, tree species mix, etc. This cultural measures can influence the expression of these trait information helps us to formulate an appropriate long-term characteristics to a greater or lesser extent. The long lifespan, silvicultural objective for the seed stand and a list of silvicul- and therefore the long lead times for provenance tests, and tural measures to achieve this objective. The necessary infor- the difficulty of organising progeny tests that best demon- mation is given in the seed stand information document, whi- strate genetic composition of the traits under consideration ch forms part of the documentation for each seed stand. This because of linked inheritance and trait characteristics that re- information is also the basis for the silvicultural plan, which is sult from the expression of a large number of genes, make it somewhat more complex and requires more information than difficult to distinguish accurately between hereditary genetic the harvested forest, where the seed production function is traits and environmental influences. The greater or lesser in- not as emphasised to such an extent. fluence of heredity on a particular trait characteristic is shown A qualitative analysis of the stand based on the in the following table. 65 Forest Seed Facilities Table 5: Relative effects of heritability and environment on trait expression in beech and oak and silvicultural measures in selected seed stands TRAIT Hereditary Environmental Effects of tending Measure influences influences straightness of the trunk low great great note tree forking great possible low remove trunk twisting great low low remove adventitious shoots low great great value vitality great great medium note trunk cracks – dry medium great medium note trunk cracks – wet low great medium remove late onset of leaves/needles great low low support thickness of branches great medium medium note approach to cylindrical form medium great great support crown length medium great great note shape of the crown great great great support damage caused by diseases and pests medium great medium note The list of selection criteria may be extended. In ा fulfilling the wood-production function of the France, it includes the physical and chemical properties of stand (seed stands are stands with a particular the wood – the thickness of the conductive elements, the quality of wood mass, and the importance of this shrinkage and tension of the wood fibres in different directi- objective is therefore emphasised); ons, the thickness of the annual ring, the colour of the wood, ा the fulfilment of all other forest functions, such as the tannin content, etc. protective and social functions (their relative im- In selection for breeding, it is necessary to decide portance is the result of their evaluation in a wider on a single criterion or a small group of selection criteria, and context). it is therefore advisable to rank them, to determine the rank Silvicultural measures (thinning) in seed stands are of each criterion for all the trees in the selected seed stand, mainly aimed at: and, in the case of silvicultural measures, to eliminate mainly ा selection for specific target traits (removing indivi-the trees with the worst characteristics, which are predomi- duals with undesirable traits, especially those that nantly hereditary. are more dependent on genetic composition); A long-term silvicultural objective determines the ा increasing seed yield (crown release); future state of the seed stand that will meet our needs. It ा maintaining adequate stand structure (distributi-should be borne in mind that a seed stand fulfils more fun- on of trees, stability, persistence of the filler layer, ctions than a normal harvested forest. Thus, the long-term slowing down of natural rejuvenation); silvicultural objective consists of several components: ा protecting genetic diversity (the size of the popula- ा the production of seed material with an excellent tion that cross-pollinates); genetic makeup, maximising genetic diversity; ा increasing the value increment of the stand (the ा the production of large quantities of quality seed wood production function is not less important, over a given period of time; only the harvesting of quality trees is delayed for ा ensuring conditions for seed collection (adequate the time when the stand will be used for the pro- stand structure); duction of quality seed). 66 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines 8.2.2 Some Difficulties in the Selection and Tending of Selected Seed Stands The selected seed stands have historically been in poor fructification even during a massive seed year for a represented mainly in conifers, with most of the seed stan- particular tree species in the vicinity of the selected stand. ds from the time of the first registers and their revisions In the past, not enough attention has been paid being approved for spruce. With the establishment of the to the development phase of the selected stand. Often they Slovenia Forest Service, the selection of seedlings for plan- have been selected during the rejuvenation phase, so that ting has shifted to broadleaved trees, and the number of they have developed a thick layer of young seedlings or seed facilities for these has been greatly increased. Howe- saplings that prevents the seed from being collected from ver, it still falls short of meeting all needs for all species in the ground. all provenance regions and altitudinal zones. The selected Since 2002, we have also been including the late seed stands are often too small, so the number of trees pol- pole stage forests in the selection of seed stands, with re- linating each other is too small. Using seed material from latively intensive measures depending on the structure and such stands, it would be possible to initiate major chan- age of the stand and the expected time of seed production ges in the genetic structure of future stands (the bottleneck of the “selected” category in each stand. In particular, we effect from the chapter on genetics). try to approve as a seed stand as large an area of forest as In the past, intervention in selected seed stands possible (optimally around 100 ha for the majority species has been very limited due to breeders/silviculturists’ fear of or a larger number of groups of minority tree species), re- losing the positive characteristics of a phenotypically supe- presenting the same population of forest trees (inter-pol- rior stand. As a result, these stands are often too thin, un- linating), in which it is possible to apply an irregular shel- tended, the canopy is cramped and, due to their age, often terwood system, leading to a mosaic structure of the parts no longer able to respond to possible release. This results of such a stand suitable for FRM production. Photo 3: A branch with cones during the mass fructification of spruce (photo by Hojka Kraigher) 67 Forest Seed Facilities 8.3 Organisation of Seed and Seedling Supply Without a comprehensive system in place to as well as seedling cultivation programmes by tree species, ensure a sustainable supply of seeds and seedlings, it is not quantity and provenance. To ensure that tree nurseries have feasible to sustainably guide forest development through an uninterrupted supply of seed, it is necessary to have planting and sowing to complement natural regeneration. transitional stocks available for years when there is no seed As a rule, seedlings are raised over several years. Therefore, crop. This role is performed by the Seed Storage. However, a medium-term programme (5-10 years) of seedling needs because of the above problems with seed production in and seed collection is needed, as a basis for planned selected seed stands, we also approve, if necessary, ordinary seedling production and, if necessary, for sowing in the seed stands or groups of seed trees (“source identified”) for forest. The medium-term programme needs to be updated the production of a limited quantity of seed in a given year. annually. On the basis of forest management plans, the As an example of the only FRM production in Slovenia, we annual reforestation programmes and the medium-term provide data on the main certificates issued (Table 6) and programme of seedling and seed requirements, annual the SFS data (Table 7) for the FRM produced in Slovenia for silvicultural plans and seed collection plans are drawn up, the years 1998 and 2018. Table 6: Seed collection in selected seed stands, ordinary stands and seed trees in Slovenia in 1998/1999 (individual lots collected and used by Slovenia Forest Service (SFS) in the same area) and 2018. TREE SPECIES Quantities needed for Quantities by certificate Seed quantity (kg) SFS in 1999 issued in 2018 Abies alba 69 69 92 Fagus sylvatica 1.316 538 Fagus sylvatica – pullings from natural 200.000 regeneration Quercus petraea 600 1.429 4.950 Quercus robur 600 1.151 7.620 Acer pseudoplatanus 136 414 90 Acer platanoides 160 Fraxinus excelsior 98 537 0 Fraxinus ornus 10 0 Prunus avium 94 30 540,5 Carpinus betulus 3 4.200 Ostrya carpinifolia 10 0 Alnus glutinosa 1 2,8 Sorbus torminalis 1 1 0 Pseudotsuga menziesii 1,3 Castanea sativa 2.600 Malus sylvestris 1,3 Pyrus pyraster 1,3 68 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines Table 7: Planting and sowing of seeds and seedlings in Slovenia in 1998 (only for reforestation after regular felling, sanitary felling is not taken into account; sanitary logging may require up to 1.3 million additional seedlings per year) and in 2018. TREE SPECIES Seed sown Seedlings (in Seedlings (%) in Seedlings in Seedlings (%) (kg) in 1000) 1998 2018 in 2018 1998/2018 in 1998 Picea abies 24 789 46 488.233 48 Abies alba 4 18 1 10.298 1 Pinus silvestris 5 41 2 5.865 1 Pinus nigra 414/32 3 1.200 < 1 Larix decidua 51 21.400 2 Other conifers 4 2.083 < 1 Fagus sylvatica 4 229 13 309.598 30 Quercus petrea 20/5 152 9 15.580 2 Quercus robur 724/1.460 68.225 7 Noble Broadleaves 353 21 Other Hardwoods 11 1 Fast-growing broadleaves 61 4 Other Broadleaves 31 5 Fraxinus excelsior N. Z./3 Acer pseudoplatanus 49.110 5 Prunus avium 21.695 2 Alnus glutinosa N. Z./0,5 17.000 2 Carpinus betulus 3.730 < 1 Populus spp. 2.805 < 1 Tilia platyphyllos 2,420 < 1 Sorbus aucuparia 842 < 1 Crataegus spp. 650 < 1 Malus sylvestris 595 < 1 Acer platanoides 575 < 1 Juglans regia 551 < 1 Castanea sativa 527 < 1 Ulmus minor 400 < 1 Pyrus pyraster 382 < 1 Sorbus domestica 38 < 1 Sorbus torminalis 27 < 1 Sorbus aria 1 < 1 TOTAL 1.710 100 1.023.830 100 69 Forest Seed Facilities 8.4 Systemic Problems in Forest Reproductive Material Production and Tree Nursery Activities (2017 Summary) We summarise the conclusions of the meeting Systemic Problems of Forest Regeneration, which took pla- ce in November 2016 at the Slovenian Academy of Sciences and Arts (SAZU). The conclusions were published in the Forestry Journal in spring 2017. Figure 13: Cover of the Forestry Journal, which published the papers from the meeting Forest and Wood: Systemic Problems of Forest Regeneration. Summary and conclusions of the Forest and Wood Scientific Meeting: Systemic Problems of Forest Regeneration. (published with permission of the Forestry Journal and proofread) On 24 November 2016, the 4th Class of Natural Sciences of the Slovenian Academy of Sciences and Arts (SAZU), the Council for Environmental Protection of SAZU and the Slovenian Forestry Institute organised the third traditional scientific meeting FOREST and WOOD, this time with the title: Systemic Problems of Forest Regeneration. In addition to academics, the meeting was attended by representatives of all key stakeholders in Slovenian forestry, representatives of the Ministry, scientific and educational institutions, planners and managers, forest owners, the Chamber of Agriculture and Forestry, the forestry cluster and forest nursery representatives. Forests are increasingly threatened by rapid climate change, resulting in extreme weather events, such as the large-scale glaze ice in February 2014, a shift in rainfall patterns and a consequent increase in pests and diseases, such as bark beetles in 2015 and 2016. It is therefore necessary to supplement the forest management system which in Slovenia has been among the most advanced – close-to-nature – in the world for decades. Unfortunately, in a context of large areas being cleared, problems of decline of individual tree species, and crown damage in areas where seed development takes place, in the reproductive part, natural regeneration cannot always guarantee successful regeneration to support all forest functions. The role of forests in maintaining biodiversity is not affected by the gradual transition through different successional phases, e.g. through the so-called silvicultural phase, but the development of wood production and some other functions is postponed to a more distant future. For forest owners, the timber industry and society as a whole, there is therefore a need to complement the existing doctrine of sustainable forest management, based on the predominan-ce of natural regeneration, with regeneration supported by planting and sowing at a higher rate than the current 3% of annual forest regeneration. This can help to speed up the transition to the wood-producing phase; the use of different tree species, which are in a minority in existing communities and do not provide adequate regeneration centres, can greatly reduce the risk of future disturbances; and the use of genetically diverse forest reproductive material can help to maintain the adaptive potential of future forests to climate change and other factors that threaten the development and the multiple roles of forests. The productive function of forests is also highlighted by representatives of forest managers, planners and owners, as well as by the wood processing industry, which has experienced a revival in recent years, as wood has traditionally been Slovenia's main renewable natural resource for boosting the economy. At the panel entitled Systemic Problems of Forest Regeneration, lecturers and other participants of the No-vember 2016 meeting FOREST and WOOD at SAZU critically addressed the state of forest seed and nursery production in Slovenia, which, due to the transition to predominantly (more than 90%) natural regeneration, has been losing traditional knowledge over the last two decades, as nurseries are being closed down and the production of planting material has 70 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines dropped to one tenth since the time before Slovenia's independence. In the wake of problems of individual tree species (ash decline, oak, alder and the already existing diseases of elm and chestnut, the expansion of bark beetle on spruce, which has been markedly accentuated in the last two years), there is a need to assess the suitability of other species and provenances of forest trees, potentially including the use of alien species (in a limited and prudent way), as well as the use of a greater number of species in reforestation to disperse the risks. Forest regeneration planning is carried out by the Slovenia Forest Service (SFS) in close cooperation with forest owners. The Director of SFS, D. Oražem, therefore stressed that climate change has placed forests and forestry in a fun-damentally different situation from the one prevailing until recently. The dilemmas are not only faced by forest owners, the timber industry, forest policy and the broadest range of forest users, but also by the Public Forest Service. The latter, through appropriate targeting of restoration works and networking, is one of the key conditions for a sustainable future forest without too many risks and for the benefit of the forest as an ecosystem, its owners, users and society as a whole. As part of forest management planning, the expert services prepare an overview of medium-term seed and seedling requirements for each tree species by provenance and altitude zone. Seed production depends on the biology of the species, as they do not produce every year, and seed storage on their biology. For example, the seed of oak trees cannot be stored for more than over one winter to maintain its germinability. The germination of our conifer seeds can be stored for decades. However, the time for raising seedlings of any tree species is always several years. Therefore, medium-term planning is important both for the adequate storage of seed stocks in the SFS seed storage and for the availability of suitable seedling forms for planting in forests. In addition to understanding the effects of the environment during seed development and seedling rearing on the physiology, phenology and success of the young forest in the later years after planting (gene expression studies – the effects of epigenetics on tree physiology), it is important to obtain seed at the time of massive fructification, when the seed is of good quality and genetically diverse, and to raise seedlings in locations that are as close as possible to the climatic conditions of the stands in which they are to be planted. Planting should be carried out in a manner that is appropriate to the climatic conditions of the forest stands in which they are to be planted. This means that we urgently need to support local or at least regionally distributed nurseries, and it is worth noting that, as Dr N. Ogris of SFI mentioned in his paper, there were at least 45 forest nurseries in Slovenia in the past, there were 16 three decades ago, and today there are only three functioning forest nurseries, while the largest forest seed-production organisation collapsed a few years ago. It is worth noting here that individual forest nurseries have for many years also financed their activities for forestry purposes from their horticultural activities, as V. Planinšek from the Omorika Tree Nursery in Muta presented with an overview of their operations. Appropriate forest seed and tree nursery planning is based on long-term and medium-term forest regenerati-on plans, using seeds and seedlings of as many forest tree species as possible, suitable for use in different provenance regions and altitudinal zones. The irregularity of fructification requires flexible financing for seed production, processing and storage, and the multiannual system of seedling rearing means that medium-term plans must provide for renewal at least five years in advance, taking account of the increasing need for reforestation following sanitary felling in the context of regular regeneration by planting and sowing. To implement these plans, the procurement system needs to be adapted for the entire plan period, and planning needs to provide for minimum, optimum and maximum levels of regeneration through planting and sowing (within the financial envelope foreseen or increased). Over the last 15 years, 25-35 different species of forest trees have been used in the context of replanting and sowing, but only a few species dominate in terms of quantity, and spruce still accounts for around 40% of all seedlings. The decision to support and plant tree species must take into account the trends in the impact of climate change on species success: T. Levanič, SFI, pointed out that the trend is negative in the lowlands and that changes in temperature and precipitation regimes are expected to lead to the collapse of lowland forest ecosystems, while in the highlands the effect is at least transiently positive. When selecting species, R. Brus and L. Kutnar pointed out that pure, large-area, sin-gle-species stands should be avoided, and as many different tree species as possible should be used to spread the risk. It is appropriate to use mainly species with which we already have experience, while at the same time starting to try out new species, which may be alien, in a more bold approach. Where there is no possibility for natural seeding, where large areas of forest are damaged, where there is a risk of erosion processes developing, or where silvicultural objectives are 71 Forest Seed Facilities not achievable due to disturbances in rejuvenation, where there is a desire to replace existing tree composition that is inappropriate to the site, or to increase the biodiversity of the stand, reforestation by planting and sowing is a necessary complement to natural regeneration, as pointed out by M. Westergren et al. (2018). Maintaining forest seed and nursery knowledge and flexible funding are important within this context. At the same time, the whole chain from seed to well established seedling planted in the forest must be improved, or, as phytopathologists N. Ogris and D. Jurc point out, in addition to ensuring healthy seedlings, it is necessary to ensure quality in a systematic way, to define the required chara-cteristics of the seedlings, the way they are dug, transported, handled before planting, the way they are planted and the way they are cared for after planting by means of a standard or a quality code, as the success of the artificial reforesta-tion by means of seedling planting is determined by all of the above requirements. In addition, regular testing for the presence of hidden, latent and cryptic pests, with a focus on Phytophthora, should be ensured in forest tree nurseries. The dilemmas are faced not only by the public forest service, which is moving towards complementing predo-minantly sustainable forest management, but also by the demands of forest owners, the timber industry, forest policy and the broadest range of users, especially nature conservation, which has traditionally been the primary focus of forest management planning. J. Krč from the Forestry Department of the Biotechnical Faculty (BFG) pointed out that careful and professional forest production (reforestation of areas affected by disturbances) has a positive impact on the success of stand rejuvenation – both in terms of the composition of mosses, herbaceous and shrub species, and the structure (composition, density, vigour and height of individual tree species) of the future stand. It is therefore necessary to create the conditions (a quality assurance system) to ensure that as much as possible of the quality reforestation of the areas affected by the disturbances is achieved. M. Humar from the Department of Wood Science at the Biotechnical Faculty (BFL) pointed out that proper planning of wood use and interdisciplinary cooperation can significantly reduce the eco-nomic damage caused by bark beetle and wood staining. If the bark beetles are removed from the forest in time, the mechanical properties of the wood will not deteriorate. On the other hand, blue stain fungi have a positive effect on permeability, making it easier to impregnate this wood with biocidal wood preservatives. In some countries, blue-stained wood is particularly valued and sought after for higher added-value products, so the emphasis in the use and valuation of such wood is on its innovative use and promotion. In addition to the biological problems, it is important to think about better organisation of the owners’ asso-ciations combining their efforts in the market and in the organisation of work in forests, given the very unfavourable ownership structure, as pointed out by State Secretary M. Podgoršek of the Ministry of Agriculture, Forestry and Food. The forest and its biodiversity do not need forestry intervention, but, as the Director of the Directorate for Forestry, Hunting and Fisheries, Mr Jakša, pointed out, in the face of such major disturbances as Slovenian forestry has faced in the previ-ous years, planned management cannot be based solely on natural regeneration of the forest, but must help nature by planting appropriate tree species. We need to ensure the productive function of the forest for the future. Forest owners cannot wait decades for natural regeneration and a century or more for an adequate composition of economically inte-resting forest tree species. At a time when we are celebrating the 30th anniversary of the IUFRO World Congress in Ljubljana, where Prof. D. Mlinšek introduced the principles of sustainable forest management to the global forestry professionals and scientists, the forestry profession is facing new challenges of climate-driven changes in forest ecosystems, which necessarily lead to action and the further development of the forest management doctrine. We are entering a phase where natural rege-neration is still the ‘alpha’, but no longer the ‘omega’ of forest management in Slovenia. We urgently need to act before we lose our traditional knowledge, while it is still possible to revive Slovenian seed and tree nurseries, and thus protect forest genetic resources, forest ecosystems and all functions of forests in Slovenia. Summarised by: H. Kraigher, A. Kranjc, N. Torelli and M. Zupančič, key notes from the presentations were provided by the lecturers and the representatives of forest tree nurseries. 72 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines SEEDLING DATA SEED AND 9 FOR SELECTED TREE SPECIES 73 Seed and Seedling Data for Selected Tree Species 9.1 European Beech (Fagus sylvatica L.) Figure 14: European beech: habitus (drawing by Marija Prelog) Beech is the most widespread naturally occurring of ecotypes and site-based races on the quality of beechnut. forest tree species in Slovenia. According to the Forest Deve- In the last period, a thesis on high-frequency electrophoto- lopment Programme of the Republic of Slovenia, in the total graphy and seed vitality, especially of beech, was written, an growing stock of Slovenian forests it could potentially com- internship thesis on testing seed vitality with tetrazolium (Ča- prise 58%, but in fact it comprises 29% of the total growing ter 1995), a PhD thesis on genetic variability of beech in Slo- stock. According to the forest inventory conducted 30 years venia (Brus 1999), and later several studies on the effects of ago (1990), it is distributed over approx. 900,000 ha (in 75% of silvicultural measures on genetic diversity (Westergren et al. all forest departments and sections), roughly evenly across 2015) and the identification of the origin of seed lots from the all altitudes, 84% on carbonate and 14% on non-carbonate 2016 seed year (Westergren et al 2017), as well as an expert bedrock, in relation to the total area of forests in Slovenia. Its study on the processing, storage and germination of beech- natural distribution in Slovenia is the result of natural expan- nuts (Finžgar et al. 2016). sion from glacial refugia and the relatively small proportion Planting of beech seedlings accounts for an incre- of planting in the history of Slovenian forestry before the asing share of replanting and sowing in Slovenia. In 1997, for establishment of the SFS. example, approximately 250,000 beech seedlings and 64,000 Research on the genetics, morphology, physiology pullings from natural regeneration were used for regular and silviculture of beech in Slovenia was mainly led by Dr planting, and more than 500,000 beech seedlings were used M. Brinar between the years 1950 and 1973. Interesting fea- for sanitary planting; in 2018, nearly half a tonne of beech- tures of the research at that time were the possibility of the nuts were used to grow seedlings, and 200,000 pullings from occurrence of a taxonomically not well defined species of natural regeneration were planted; in total, beech seedlings Balkan beech or Moesian beech (Fagus moesiaca (Domin, accounted for almost one-third of all seedlings planted. Re- Maly) Czecz.) in Slovenia, whose characteristics are related forestation by planting and sowing beech is constrained by to, among other things, the rooting ability of cuttings; he irregular harvesting and the high cost of storing the beech- also described a beech population with oak-like bark, and nuts for a long period, which affects the difficulty or delay in another beech population with birch-like bark. Within the implementing silvicultural plans. Below are some basics on context of seed and germination physiology, the emphasis the physiology of flowering and fructification of beech tre- was then on research into the effects of cholines on germi- es, as well as the problems of storage, dormancy and the nation and alternation of forest tree species, and the effects planning of a coordinated germination of beechnuts. 74 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines 9.1.1 Flowering and Fructification Beech trees reach reproductive maturity between the ages of 60 and 80, and solitary trees between the ages of 40 and 50. It blossoms at about the same time as it develops foliage, from early April to mid-May. The flowers are monoe- cious; separate male and female flowers grow on the same tree. They are sensitive to frost. The beech is an anemophi- lous plant, where each pollinated female flower produces a single fruit, the beech fruit, where 2–3 (4) together in a prickly cupula form a beechnut. The beech seed (a fruit, in fact) ripens in Septem- ber and October. beechnuts continue to fall down till the end of November. Wind and rain accelerate the release of the nuts. The small triangular-shaped pyramids are about 1.5 cm long. The pericarp is relatively thick, smooth and shiny. As the seeds dry out, they change colour from dark to light brown. Ripe beechnuts have a moisture content of 20-30%, depen- ding on weather conditions. It does not have any endosperm. The contents of the seed are filled by two twisted cotyledons and an embryo axis. The seeds are edible. Beechnut production starts in stands usually at the age of 70/80 years and can last up to more than 200 years. Seed production is irregular, depending on microclimatic conditions. A full crop can be expected every 5-10 years and an incomplete crop every 3-5 years. In the intermediate years, either there are no seeds on the tree, or individual seeds develop, but they are mostly empty. The first seed that falls in September is usually of poor quality, empty or contains parasites – insects. Some of the characteristics of beech mast are shown in the following table. Table 8: Characteristics of beechnuts (1 - taken from Suszka, Muller, Bonnet-Masimbert 1996, 2 - taken from Regent 1980) Number of beechnuts per kg – when harvesting (humidity 25%)1 3,000–5,000 – after drying (humidity 8%)1 3,500–5,800 – when harvesting 3,600–6,800 (humidity 22-30%)2 (AVG. 4,600) Weight of 1,000 nuts 150–300 g 1 (avg. 250 g) approx. 50 kg of Weight of 1 hectolitre of nuts1 fresh beechnuts, Figure 15: European beech: stages of female and male flowering 39-45 kg of dry beechnuts and the fruit of the European beech tree (drawing by Marija Prelog) 75 Seed and Seedling Data for Selected Tree Species 9.1.2 Harvesting and Processing of beechnuts Beechnut is almost always harvested from the ा in a water settling tank to remove empty seeds; ground. If closed fruits are collected from fallen trees, they ा in the air blower to remove light impurities; should be spread out in a thin layer in a dry place until they ा in an air screen, in which different mesh sizes (e.g. open, then the individual beechnuts are separated by sha- upper with 9 mm openings, lower with 8 mm ope- king and sifting. It is recommended to harvest immediately nings) are used to remove stones, small beechnuts after they fall off to minimise losses to rodents and birds and and light impurities in jets of air. to reduce the development of diseases. Clearing the ground By foreign standards, the acceptable purity of a lot under the trees improves the conditions for picking. It is not of beechnuts is 85-95%. recommended to harvest the beechnuts that fall off first. Beechnut is an orthodox seed, which means that Hand-picking is the most widespread method. its moisture content can be greatly reduced before storage Other methods include the use of nets and punctured foils, without affecting its vitality. The degree of drying depends sweeping under trees and vacuuming with portable vacuum on the intended storage time and the drying method used, cleaners. In each case, a mixture of fruits, beechnuts, twigs, but in any case the temperature during drying should not leaves and stones are collected. The first cleaning can be exceed 18-20°C. done in the field, removing stones, sand and most of the fo- When stored over one winter, the beechnuts may liage. The beechnuts are then spread in a thin layer for a few be dried to 20-25% moisture and stored at 3°C in a dry weeks near the harvesting site. Each lot is spread separately substrate (peat or vermiculite) or dried to 12% moisture and on the ground to partially dry. During this time, the beech then stored at 3°C until the end of the winter, when a mo- mast is hand-mixed on a daily basis. Another way of sorting ist pre-treatment at the same temperature is started before and cleaning is by using water settling tanks, but in this case germination. When storing for more than one year, it is es- immediate partial drying of the beechnuts is necessary. sential to reduce the humidity level to 8-9% before the start The mixture of beechnuts and impurities is tran- of storage, which takes place at -5 to -10 °C in hermetically sported in jute sacks or nets to the processing site. In Slove- sealed containers. It can be dried at 18–20 °C with a fan, a nia, processing was carried out at Semesadike Mengeš until procedure that can be interrupted by drying without blowing. their closure, and later at the Omorika Tree Nursery in Muta Drying can take from a few hours to a few days, depending and at the Slovenian Forestry Institute for the needs of the on the relative humidity and moisture content of the seed seed storage of the Slovenian Forest Service. On arrival, the lot. Drying capacity can be improved by using dry air by con- moisture content of the beechnut is usually between 25-32%, densing the moisture on the surface of the condenser whe- therefore it is dried first (48 hours in the air or in an oven at re the cold water circulates or on the surface of the cooling 18-20 °C). It then goes on to mechanical cleaning, depending system in the refrigerator. After drying, the beechnuts must on the type and amount of impurities: be cleaned again. 9.1.3 Storage of Beechnuts, Dormancy Breaking and Germination There is a distinction between short-term storage break dormancy. over one winter (until spring sowing) and long-term storage due Beechnuts can also be stored in a cold pit, but in this to irregular periodicity of fructification (up to five or six years). case a large amount of water must be added three weeks be- Short-term storage can take place with or without fore sowing and then the beechnuts must be stirred daily until medium/substrate. The choice of method depends on how sowing. Sowing follows when the seed root exceeds 1–2 mm in the dormant beechnut is removed before sowing. Traditional length. storage methods are based on maintaining humidity above Beechnuts that desiccate below 20–25% can also be 25% during the storage period, possibly with the help of water stored in a dry substrate (vermiculite or sand). The mixture is sprinkling nozzles and by including a cold period (but without then stored in a refrigerator (3–4 °C) and the substrate is mois- freezing). Thus, beechnuts (without substrate) can be spread re- tened several times before sowing to break dormancy. In addi- gularly on bare forest floor and covered with a layer of straw or tion, in France, beechnuts are stored at 12% moisture in herme- leaves, and later with a blanket of snow. The gradual melting tically sealed containers at 3 °C, followed by pre-treatment with of snow causes imbibition of the seeds, which is necessary to or without substrate. 76 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines It is difficult to determine the degree of dorman- including storage at elevated C02 concentrations. cy and therefore the pre-treatment time for all these pro- Determination of seed dormancy (X) for beech: cesses, as a lower dormancy develops during the storage On a sample of 25 seeds (for each individual lot), the dor- period, especially when the moisture content is above mancy rate (X) is determined by a germination test on 10% 20%. It is therefore advisable to use the same method for vital seed (seed root development) at 3 °C (on filter paper short-term storage as for long-term storage, as this ma- or wet vermiculite in a cold chamber) at a humidity of 30- kes it easier to determine the degree of dormancy and the 32% (never up to 34%!); the vitality of the seed is determi- pre-treatment time of the seed. ned by a TTC test. The X time can vary from four to a maxi- Long-term storage is necessary due to the irregu- mum of 20 weeks, depending on the seed lot (provenance, lar beechnut production. Successful methods have been year of harvest, method and time of harvesting, etc.). developed in a collaboration between France and Poland, Cold stratification of beechnuts A) Cold stratification of the seed in the medium: ा the seed is treated dry with a fungicide (against the fungus Rhizoctonia solani J.G. Kühn); ा then it is fully moistened by contact with a moist medium (not in excessive wetness); ा then it is stored in a moist medium at 3 °C until the seed root appears in 10% of all vital seeds; depending on the X, this period may last from 1 to 3 months; the time of stratification cannot be extended, although it may last for approx. 50 days for all the seeds to germinate. B) Removing dormancy without medium by pre-cooling: ा the seed is moistened to 30-32% (never 34%!) at 3 °C; ा followed by a fungicide treatment (wet); ा Then it is stored at 3 °C at a humidity of 30-32% (never 34%!) for X + 2 (or X + 4) weeks; the X time depends on the degree of dormancy of the seeds and is important for further storage (the X time can vary from 4 to a maximum of 20 weeks, depending on the seed lot); a constant time of 6-10 weeks can also be used; ा since the humidity level is sufficient to break dormancy but not to germinate, it is possible to completely break dormancy of all seeds before germination in the tree nursery or when conducting a germination test. In each case, the seed is placed in small covered 40-80 cm tall seedlings are used, which require 3-5 years of trays on which the date of the start of the process and the cultivation in a tree nursery (1 + 2 or 1 + 3). In France and Po- estimated date of completion of the process are written. The land, non-dormant seed is usually sown at the end of April trays are placed in a cold chamber and periodically checked or beginning of May, 5 to 7.5 kg of seed per 100 m2 bed in for moisture and infection (once every one to two weeks, at rows, whereby they cultivate 8,000 to 32,000 seedlings per least once a week at the end of stratification). In case of in- 100 m2. The covering layer is 2-3 cm thick. Fungi of the genera fections, the infected seed should be removed and the rema- Phytophthora, Pythium and Rhizoctonia usually need to be ining seed washed and placed in a new medium. suppressed. By sowing in tunnels, it is possible to cultivate Seed quality analyses: Seed testing is carried usable seedlings in just one year. In any case, the soil quality out according to the general ISTA protocols. The size of the needs to be adequate to allow good root development. sample taken is 1000 g and the size of the working sample is 500 g. Due to dormancy, the vitality test usually replaces THE SOURCE OF THE ILLUSTRATIONS: the germination test. Analyses of cut seed can be used, with Manual for Forest Genetic Monitoring the TTC test being the most common one, while radiography Bajc, M., Aravanopoulos, F., Westergren, M., Fussi, broken, tests of emergence of germlings are used in additi- 2020. Priročnik za gozdni genetski monitoring. / Manual for Forest Genetic Monitoring. Ljubljana: on to germination tests to provide better information on the gives less information about the seed. After dormancy has B., Kavaliauskas, D., Alizoti, P., … Kraigher, H. (ed.). Slovenian Forestry Institute, Silva Slovenica germination capacity of the seed lot. Publishing House. http://doi.org/10.20315/SFS.168 Sowing in the tree nursery: For forestry planting, 77 Seed and Seedling Data for Selected Tree Species 9.2 Oaks: Pedunculate Oak (Quercus robur L.) and Sessile Oak (Quercus petraea Liebl.) Figure 16: Oaks: habitus of pedunculate (left) and sessile oak (right) (drawing by Eva Margon) In Slovenian forests, oaks account for 8% of the and morphologically distinct populations of all three speci- growing stock. Six or seven species occur naturally: Q. robur es. Elsewhere, especially on intermediate sites between oak L. (7% of the total oak timber stock), Q. petraea (Matt.) Liebl. species optima, the differentiation process is not complete, (82 %), Q. pubescens Willd. (2%), Q. cerris L. (8%), individual resulting in a multitude of intermediate, hybrid oak ecotypes. Q. ilex L. and Q. crenata Lam., and there are also records of Q. The occurrence of hybrids limits the accuracy of the forest in- virgiliana, which later turned out to be a hybrid of downy oak ventory data, so that a knowledgeable forester can also record with sessile and peduniculate oaks (Jerše and Batič in Gozd V the thriving of pedunculate oak at high altitudes and on hill 2007). The introduced species are Q. rubra L. and Q. palustris. stands (above 1000 m), and hybrids and single pure speci- The annual harvest of all oaks is in the order of magnitude of mens of sessile oak can be observed on transitional sites in 90.000 m3. the Krakov Forest. Lowland oak forests are among the most altered The annual number of planted oak seedlings is forest ecosystems in Slovenia. Most of them have been 150,000 for the two major species (about 9% of the total), and converted into farmland or built up. The oaks’ plot is thus highly the quantity of acorns varies from about 300 kg to several fragmented, with small, isolated populations, often under the tonnes per year for each species. Around 100 to 300 kg of constant influence of pollution and changes in groundwater Turkey oak and downy oak seeds have been collected and levels. The largest complex of pedunculate oak forests is in the sown to restore the Karst burned areas. area of the intermittently flooded Krakov Forest, which covers about 3,000 ha. Sessile oak also thrives on more arid sites, mostly up to 700 m above sea level, occasionally above 1000 m, but this should also be checked due to possible mistakes with pedunculate oak done by inventory-takers. The group of systematically related oak species – pedunculate oak, sessile oak and downy oak – in Slovenia is characterised by high genetic and morphological diversity, frequent interspecific hybridisation with introgression of ge- nes from one species into another, and undefined ecological niches of individual oak species. The source of species di- versity in oaks is their genetic system. In evolutionary terms, the development of the oak trees studied today is at different stages. In some environments, the differentiation of peduncu- Figure 17: Oak: leaves and fruits of the pedunculate oak (drawing late, sessile and downy oaks is complete, resulting in separate by Eva Margon) 78 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines Pedunculate and sessile oaks reach reproductive maturity in the stand around the age of 50-60, and as solitary trees at around 30-40 years. The optimum age for producing quality seed is between 100 and 220 years. The oaks are mo- noecious and pollination is by wind. They blossom in May and June. Of particular interest in the case of pedunculate oak are the late-flushing individuals and populations, which are of interest from the point of view of resistance to spring frost and pests. Flowering can be relatively regular every few years, but seed production is highly dependent on weather conditions, so a good fructification is not common. The acorn grows to its full size by the end of the au- tumn. The first acorns start to drop in the summer in case of drought, and in August and September there is usually a high proportion of empty seeds and seeds that become home to pests. Acorns of both species usually reach physiological ma- turity and adequate quality in October. The following table Figure 18: Oak: leaves and fruits of the sessile oak (drawing by provides information on acorns. Eva Margon) Table 9: Characteristics of pedunculate and sessile oak acorns (1 - taken from Suszka, Muller, Bonnet-Masimbert 1996) Pedunculate oak1 Pedunculate oak Sessile oak1 (France, Poland) (Krakov Forest) (France, Poland) Number of acorns per kg 145–500 (avg. 250) 130–650 (avg. 375) Weight of 1,000 acorns 2–7 kg (avg. 4 kg) 4,6–7 kg (avg. 6 kg) 1,5–5 kg (avg. 3.1 kg) Weight of pure acorns per hectolitre 60–80 kg 50–80 kg Acorn mass per tree (120 years, crown ~ 20 kg with 120 m 2 , strong mast production) Acorn weight per ha (good yield) 1–2 t/ha Because of the natural cross-fertilisation betwe- and handling them. When harvested, the seed is mostly en the pedunculate and the sessile oak, it is advisable to 90% pure. It is stored and transported in thin layers and carry out morphometric analyses of the foliage in the li- jute bags. tterfall. By multivariate analysis of at least five leaf cha- The seed is not capable of drying (recalcitrant) racteristics, it is possible to determine the taxonomic and its moisture content must not fall below 40-42%. Be- affiliation of each tree with relative precision, and the cause of the high physiological activity, the temperature population mean. In principle, only a pure stand of one of the seed can rise rapidly during storage and transport, species may be approved as a selected stand. while the humidity drops. The acorn often carries spores Acorns are harvested by hand from the ground of the pathogenic fungus Ciboria batschiana Buchw., whi-in October. An individual worker can collect between 20 ch mummify it. For this reason, the seed is usually sprayed and 50 kg per day. Nets can be spread in the stand before with benomyl and similar preparations (0,4 g/kg acorn) harvesting, or they can try to find the caches of squirrels and, in larger trees, thermotherapy (two to two-and-a-half that collect the top-quality acorn. Acorns are sensitive to hours in water at a temperature of exactly 41 °C) is also impact shocks and care must be taken when collecting applied before storage. Seed is difficult to store, and usu- 79 Seed and Seedling Data for Selected Tree Species ally remains vital at high humidity (40-45%) at around -1 °C Tree nurseries in France produce around 7,000 se-for only one winter or up to a maximum of 18 months (up edlings per 100 m2 bed from around 35 kg of acorns. The to three winters in small quantities in the laboratory). Se- sowing density in France is around 25 acorns per linear me- veral research groups in Europe are working on developing tre. In Poland, about 55 kg of acorns are sown in rows in the a method to store acorns for longer, but so far without su- same area in the nursery, and 250-300 kg are sown undis- ccess. Acorns are not dormant and can sprout in autumn. tributed in the bed. It is covered with a 3-5 cm thick layer Postmaturation and precalcification of the acorn are part of soil or other material. The germination rate in the tree of the research on the development of a method to store nursery is lower than in the tests: the normal germination young seedlings or isolated embryos. rate for acorns of the 44% humidity period is up to 100%, Seed quality analyses: The sample size is 2.5 kg with 37% humidity it is 89%, while acorns from the Krakov for the test sample, 1 kg for the working sample, including Forest germinated at 82% at 37% humidity. The average ger- approx. 500 acorns For germination tests, 1/3 of the acorn mination rate in an open nursery in Poland is around 50% is cut off and the testa removed using the ISTA method. for pedunculate oak and 70% for sessile oak. Figure 19: Oaks: stages of male flowering (drawing by Eva Margon) 80 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines 9.3 European ash (Fraxinus excelsior L.) The European ash is a widespread species that thrives from lowlands to altitudes above 1000 m. It thrives in approximately 120,000 ha of forests, mostly in small grou- ps or individually. For reforestation by planting and seeding until the appearance of the ash canker (Chalara fraxinea, Hymenoscyphus fraxineus (Baral et al. 2014), Chalara fraxinea (Kowalski et al. 2006, Hymenoscyphus pseudoalbidus (Queloz et al. 2011) an average of 200-400 kg of seed was collected annually and around 100,000 seedlings were planted, or 5% of the total number of seedlings to be planted during regular cultivation work. After 2010, due to the recommendations of phytopathologists, ash seedlings are practically not used in tree nurseries and for planting in forests, although it would be appropriate for the conservation of the species to syste- matically propagate more disease-resistant specimens and to support the increased planting of European ash seedlings with financial incentives, as this would be the only way to conserve this species (and the related species) in our forests. The biology of flowering is complex, as usually one of the two sexes of flowers on a tree dies off, resulting in male or female trees, which may also differ in growth phenotype. The ash reaches reproductive maturity at the age between Figure 20: European ash: habitus (drawing by Metka Kladnik) 30 and 40 years. It flowers regularly every two to three years. a) b) c) č) d) Figure 21: European ash: stages of flowering, female and male flowers. a) bisexual (top), male (left), female (right), b, c, d, e) male flowers, d) female flowers (drawing: Metka Kladnik). The samaras, which are attached in clusters, gra- green seed takes place at the end of August. Green samaras dually develop until the end of September or October. They cannot be stored, they are immediately used in sowing. Ash are still green in August, then gradually dessicate on the needs a few months of warm stratification followed by a few tree and turn brown. Samaras are about 4 cm long and 6-8 months of cool stratification. Samaras sown while they are mm wide, each containing a single seed. The embryo deve- still green have adequate time to produce embryos before lops in the middle of the endosperm to about half its final the onset of the cold season. The timing of the first warm size at seed maturity. period is very critical; if it is too short, the success of sowing Characteristics of the large ash samaras: the weight green samaras is poor, the seed will survive and germinate of 1,000 samaras is 65-100 g, the number of samaras per kg is only in the second spring. It is therefore better to collect 10,500-15,500 and 1 hectolitre of samaras weighs up to 15-20 kg. brown seeds. Seeds collected in October and November The harvesting of the mature seed usually takes should be stored in the tree nursery and sown after pre-tre- place in October and November, while the harvesting of the atment in the second spring after collection. 81 Seed and Seedling Data for Selected Tree Species During harvesting, the samaras can have a humidity ा TTC: the seed is removed from the samara and soa-of around 50-60%, therefore they must be dried at a tempe- ked in water for 18 hours; it is then cut 0.5 mm along rature below 20 °C in a thin layer to a humidity of 8-10%. The both sides and soaked again for 8 to 24 hours at a seeds can be dried and can be stored for a long time at -3 to temperature of 30 °C in the dark in 1% TTC at pH 6.5- -5 °C for up to 10 years. Shorter storage is possible in a venti- 7.5 (fresh seed is soaked for only 8-10 hours); after lated, shaded shed in jute sacks. rinsing in water, the endosperm is cut in half and Breaking of dormancy: The complex form of dor- the embryo is examined, which must be completely mancy requires both warm and cold stratification, in or out stained; only minor necrosis in the endosperm far of the medium. from the embryo is allowed; In the media: The seeds are completely moiste- ा indigo-carmine and X-radiography are less frequen-ned in contact with the medium (peat:sand in a 1:1 ratio); if tly used for ash. necessary, they may be treated with a fungicide (in France, In the tree nursery, green samaras are often sown in copper oxyquinoleate); the moisture content of the medium late summer, but success rate can vary widely. It is recommen- has to be checked weekly. ded to sow the stored seed from March to April after using No medium: The seed is gradually moistened by various dormancy-breaking methods. It is important that the spraying to 55-60% moisture (at this moisture dormancy is warm period lasts about 16 weeks. Cover the seed with 1-2 cm terminated and the seed has not yet started to germinate). It of sand. In Poland, 2-3 kg of samaras are sown in rows in 100 is then treated with a fungicide and stored in a plastic conta- m2 beds, producing about 8,000 seedlings or up to 30,000 se-iner which must not be hermetically sealed (limited air circu- edlings per bed. The sowing rate of vital seed per m2 is 40-60, lation must be permitted). The humidity level is determined with a yield of 30-50 seedlings. 160-200 vital seeds are sown in weekly by weighing. plastic tunnels for cultivating up to 80 seedlings. Procedure: First, the seed is stratified in a warm pla- ce for 6-16 weeks (usually 16 weeks) at 15 °C or 20 °C (emb- ryo growth time in the seed until it fills 80-90% of the seed length; it can be inspected by in longitudinal section under a magnifying glass), then 16 weeks at 3 °C (removal of embryo dormancy). Seed storage: After stratification, the seed can either be: ा immediately sown (or germination is tested by the ISTA method or according to the French method at 5-15 °C (14 h + 10 h) and the Polish method at 3-20 °C (16 h + 8 h) for 8 weeks on filter paper; ा stored damp, non-dormant at -3 °C for up to 8 we- eks; ा gradually dessicated at around 20 °C to 8-10% mo- isture and stored non-dormant for two years or more at a temperature of -3 °C to -5 °C. Seed quality analyses: The ISTA test sample weighs 400 g, the working sample 200 g. For dormancy reasons, only vitality tests shall be carried out: ा in longitudinal section after soaking in water for 18 hours; used for % of full, empty, parasitised and dead seeds, usually for a quick test before harve- sting; vitality is indicated by a white embryo in a white endosperm, but must be verified with vitality Figure 22: European ash: leaves and seeds (samaras) (drawing by staining methods; Metka Kladnik) 82 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines 9.4 Norway Maple (Acer platanoides L.) Norway maple is widespread at different altitudes, humidity from an average of 35% to 11.5%. After the second growing singly or in small groups. It is rarely planted in fo- week of drying, it drops to 8%. restry, so it is shown mainly for comparison between ortho- Storage: The samaras of Norway maples are rarely dox and recalcitrant seed of the same genus. stored because the seed years are quite frequent. If stored, It reaches reproductive age at 40 years in the stand they are kept at a humidity of 8-10%, in closed-off conta- and 20 years outdoors. It blossoms in April and May; its iners, at a temperature of -3 to -10 °C. The seeds are re- flowers are bisexual, pollinated by bees. The seeds consist sistant to low temperatures even at high humidity. of two samaras, which are grown at an obtuse angle of more Removing dormancy: Cold stratification at a tem- than 90°. The two samaras separate before they are disper- perature 1 or 3 °C for 6-14 weeks in a moist medium is used; sed. The seed is brown at maturity, has no endosperm, and in this case, the seed germinates best after the end of stra- the embryo is completely green even when dormant. The tification, also at a temperature of 3 °C. Cold-warm stratifi- seeds ripen in September and the samaras are dispersed in cation and cold-warm germination are also possible, but in October and November by the wind. The seed year is either this case germination may be greatly reduced with minimal every year or at least every two to three years. irregularity; freezing the seed at a temperature of -3 °C for Characteristics of Norway maple samaras: weight 0-20 weeks prior to germination is also recommended, with per 1000 samaras is 100-400 g (avg. 140 g), number of sama- the temperatures gradually rising from 1-3 °C, 1-5 °C, 1-10 °C, ras per kg is 2,800-10,300 (avg. 6300). 1-15 °C, 1-20 °C to a constant temperature of 20 °C. Seed vitality is usually in the order of magnitude of 75%. Harvesting takes place in September, before the sa- maras turn brown. After this time, they are easily dispersed by the wind. A single tree can produce up to 15 kg of sama- ras in a seed year. The weight of seeds that a worker can collect per day from a single tree is 2.5-5 kg. In the stand, harvesting is much more tedious. Drying: Green seed (early October) usually has a moisture content of 49-59%; brown seed (late October/early November) 32-41%. The seeds are dried at a temperature of 17-19 °C in a dry place for about one week to reduce the Figure 23: Norway maple: seed (drawing by Teja Milavec) Vitality test: 1. By cutting (only for fresh seed material): 1) the seed is removed from the coat; 2) empty and damaged seeds are counted; 3) the rest of the seeds are cut; the problem is green cotyledons, which can be green even if the seed is dead; 4) a live seed is more likely to be one whose cut surface glistens, the cotyledons are imbibed and the seed is not damaged in the slightest; 5) if this test is carried out on dry seed or seed that has been stored for a longer period of time, the results can only be expressed as % of full seeds. 2. TTC: 1) 4 × 50 or 4 × 100 seeds are soaked in water for 18 hours; 2) the pericarp is removed; 3) a small part of the seed coat is removed and the seed is once again soaked in water for a few hours until it is completely imbibed; 1) up to 3): dry seed or seed that was stored for a longer period should instead be cold stratified at a temperature between 3 and 5 °C for 10-14 days; 83 Seed and Seedling Data for Selected Tree Species 4) the naked embryos are then soaked for 5-8 hours at a temperature of 30 °C in a 1% TTC solution in phospha- te buffer (pH 6.5-7.5); 5) vital seed is seed that is fully coloured or where the radicle is not coloured or where there is minor necrosis on the distal part of the cotyledons; 6) non-vital seeds are those with visible necrosis near the embryonic axis. Germination test according to the ISTA method (1996): ver, this can lead to premature germination in mild and wet ा cold stratification (freezing) in a moist medium winters. If the samaras are dried, they must be stratified (sand and fine peat) at a temperature between 1 in a cool place from May to autumn, the humidity of the and 5 °C for two months (seed wings are cut off); medium is increased at the end of autumn, and the seed conduct weekly inspection; is sown in spring of the following year. Seeds often start to ा then germination test on sand or paper for 21 germinate before sowing and even a drop in temperature days at a temperature of 20 °C. below 0 °C does not stop this process. In the tree nursery: The seedlings of Norway Storing the samaras at approximately 10% humi- maple can be sown immediately after harvesting (between dity in closed containers at a temperature of –3 to –10 °C 15 September and 15 October) in the tree nursery, or after is recommended. After thawing, they may be stratified at short-term storage of partially dried seeds, or after drying a temperature of +3 °C for a period to be determined on and short- or long-term storage at low controlled tempera- the basis of a control sample in stratification. The sowing tures. After sowing, they are covered with 2-3 cm of soil or a time can be calculated from the information in the con- mixture of sawdust and sand. Sow 4 kg of samaras in rows trol sample. In this process, premature germination can be per 100 m2 bed or twice that amount over the entire bed. stopped by lowering the temperature to -3 °C for up to 8 Germination is usually in the range of 50-90%. This bed can weeks. All stages of this seed processing are carried out grow 10,000-30,000 seedlings. under controlled conditions, independent of external fa- Samaras can also be stored in the forest over win- ctors. ter, covered with leaves or in pits, mixed with sand. Howe- 9.5 Sycamore (Acer pseudoplatanus L.) Sycamore is a widespread species. It grows at all grown at an angle of 60-90 °C. The samaras are 36-55 mm altitudes singly or in groups in mixed stands. In Slovenia, it long and each contains one pea-sized seed without endos- is most common on carbonate soils, although it also thri- perm. Samaras ripen in autumn, when the wings turn light ves elsewhere, especially in the altitudinal zone from 400 brown exhibiting darker veins. to 1000 m. Seeding of sycamore occurs regularly every two to It blossoms from April to May, before and during three years, it is abundant, from the age of 20 in the outdo- leaf-out. The flowers form clustered inflorescences that can ors and from the age of 40 if grown in a stand. be 13-20 cm long. The flowers are bisexual, but usually either Characteristics of sycamore samaras: weight per the stamens or the pistils deteriorate, so that they can only 1000 winged samaras is 66-180 g (average 120 g), number function as male or female flowers in different proportions of winged samaras per kg is 5,500-16,000 (average 11,000) of the two sexes. In the inflorescence, the basal and central samaras. flowers are usually female and the terminal flowers male. Collecting: On dispersal, the two samaras separate Monoecious inflorescences are rare, as are monoecious into independent fliers. The wind can blow all the samaras trees. Flowers of different sexes mature at different times, off a single tree in a single day. with a flowering period of 7-15 days. Pollination is mainly Harvesting takes place before the windy season, by insects. from September to mid-October, when the samaras change The fruits are clustered in clusters up to 29 cm colour from green to yellow. They need to be sown or stra- long, with the number of fruits per cluster varying from a tified immediately and may germinate prematurely. Seeds few to more than 30. Each fruit consists of two samaras, can be collected on the ground or from the tree, by shaking 84 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines or climbing, or from felled trees. seed and air humidity. If the seed starts to germinate, it is The leaves and stems of the clusters are removed stored in peat at a temperature of -3 °C. by preliminary cleaning in the field. They are transported For longer storage, the seed is dried to 24-32% in bags with large openings to prevent the development of moisture, stored in closed containers at a temperature of mould. The final cleaning is done by sieving or blowing. The 0 °C for one day, then at a temperature between -3 and -5 wings may remain on the seed. °C for the entire storage period. Drying: The moisture content of samaras during harvesting is 42-55% of the fresh weight. Sycamore seed cannot be dried (recalcitrant) and the moisture content must not fall below 24% (for whole samaras). When storing for one winter, drying is not necessary, but for longer pe- riods, the seed with wings is dried to 24-32% moisture or the pure seed to 30-42% moisture with air circulation at a temperature between 18 and 20 °C. Drying under natural conditions is difficult in October and November because of the fluffy layer that insulates the pericarp seed. Storage: The samaras can be stored for one win- ter in moist peat at a temperature of 3 °C and 40-50% Figure 24: Sycamore: seed (drawing by Teja Milavec) Removing dormancy: ा In the medium (5-14 weeks, exceptionally up to 20 weeks): ◊ after fungicide treatment, the seed is moistened in a moist medium (peat, sand or a 1:1 mixture); ◊ then stored at a temperature of +3 °C until 10% of the seeds have germinated (usually for about 14 weeks); such seeds can be sown immediately or stored at a temperature of -3 °C until germination; ◊ at a temperature of +3 °C germination is very slow (8-12 weeks). ा No medium: ◊ the seed is gradually moistened to 44-50% (whole seed) or 50-58% (without wings); ◊ this is followed by fungicide treatment; ◊ the seed is then stratified in a thin layer at a temperature of +3 °C in plastic containers, lightly covered with plastic film (not sealed); weekly humidity inspection; ◊ the time for stratification is determined by the dormancy rate (X time – same as for beech); ◊ after stratification without medium, the seed can be stored for two winters at a humidity of 24–32% at a temperature of -5 °C in closed containers. Vitality test: ा By cutting (3 × 50 or 3 × 100 seeds); the problem is green cotyledons, which can be green even if the seed is dead; the result can only be expressed as % of full, empty and parasitised seeds. ा TTC: 1) the seed is cold stratified between temperatures of 3 and 5 °C for 10-14 days; 2) the pericarp and part of the seed are removed; 3) the seed is then once again soaked in water for a few hours; 4) the naked embryos are then soaked for 5-8 hours at a temperature of 30 °C in a 1% TTC solution in phospha- te buffer (pH 6.5-7.5); 5) vital seed is that which is fully coloured or in which the radicle is not coloured or there are minor necroses 6) at the distal part of the cotyledons; 7) non-vital seeds are those with visible necrosis near the embryonic axis. Germination test according to the ISTA method (1996): ा cold stratification (freezing) in a moist medium (sand and fine peat) at a temperature between 1 and 5 °C for two 85 Seed and Seedling Data for Selected Tree Species months (seed wings are cut off); conduct weekly inspection; ा then germination test on sand or paper for 21 days at a temperature of 20 °C. Improvements of the germination analysis: ा after cold stratification (7-20 weeks), it is better to carry out the germination test at a temperature of +3 °C; ा after cold stratification, germination can also be determined by altering the temperature during the germination test at temperatures between 5 and 15 °C (3-4 weeks is sufficient for complete germination of the seed); ा the emergence test conducted in the tree nursery: 4 x 50 seeds pre-treated in medium until germination; the wings are then cut off and the seed is planted in containers with moist medium at temperatures between 3 and 20 °C (16 h + 8 h day) for 12 weeks (emergence is observed on a weekly basis). Sowing in the tree nursery: Sowing fresh seeds can be done immediately after harvesting in autumn. Stratifica-tion is necessary when sowing stored seeds. In Poland, 3.5 kg of samaras are sown per 100 m2 bed in rows or 8-10 kg over the entire area. This can produce 10,000-30,000 seedlings. The sowing density is 160-300 samaras per m. The crop needs to be protected from birds and rodents, sometimes against frost, and should usually be kept shaded. In France, seedlings are grown in plastic tunnels on fertilised peat for a year. The sowing density is about 160 samaras per m2, with an average sowing success rate of 50%, which results in about 80 seedlings per m2. In Poland, the figures are twice as high. 9.6 Black Alder (Alnus glutinosa (L.) Gaertn.) Black alder grows in Slovenia mainly in the lowlands and up to 700 m, but it rarely occurs at an altitude above 1000 m. They are more abundant on carbonate soils, especially along watercourses, and on wet soils. The flower buds burst in the first year, followed by flowering, pollination, fertilisation and fruit ripening in the following year. Plants are monoecious. They bloom before leaf-out, between February and April. The male flowers hang from the ends of the branches like catkins, and the female flowers are clustered in two to three cones that develop at the base of the twigs, where they grow alongside the previous year's cones. The new strobili are characterised by a distinct stalk, 1.5-2.5 cm long. Pollination is by wind, in spring, but fertilisation only occurs in summer. Figure 25: Black alder: cones (drawing by Teja Milavec) The seeds ripen in “cones” in September or Black alder seed characteristics: 1000 seeds weigh 0.7-1.5 October. The strobili become woody and can remain on g, 500,000-780,000 seeds per kg, the weight of pure seed the tree for a long time. They open after the first hard frost in 1 hectolitre of cones is 1.5 kg, the weight of 1 hectolitre of winter, and the seeds are dispersed until spring. The of cones is 30 kg, the weight of seed from 100 kg of cones fruit is a winged nut containing a seed without endosperm. from a seed plantation in Germany was 9-19 kg. Seed germination is highest when harvested in October, Mature fresh seed has a moisture content of but declines when harvested later. 8-9%. It can be carried by the wind in a radius of 30-60 Trees in stands start to set seed at 30 years, in the m around the tree, but it can also be carried by the water, open at 15. They can go to seed every year, but only every because its wings allow it to float on the water. It can two to three years will they produce a strong crop. Seed germinate even after 12 months. set is strongly influenced by the weather at flowering time Natural rejuvenation is successful on bare soils and also a year earlier, when the flower buds are forming. or in a humus layer, free of competitive weeds and other 86 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines vegetation, under favourable moisture and light conditions. analyses is 8 g and the weight of the working sample is 4 Germination is on average around 40%, reduced by g. The vitality analysis can be carried out by means of seed broken cone scales and fungal infestations. The cones are X-radiography, which provides information on full, empty, harvested in November and December. Around 15-20 kg of parasitised and damaged seed. Only full seeds can then cones need to be collected to obtain 1 kg of seed. In a be selected for germination analysis. The germination good harvest, one worker can harvest up to six kg of cones. test is carried out in a Jacobsen germinator, where the Because of the high humidity, the cones have to be quickly temperature is raised to a temperature of 27 °C once a day transported to the seed factory for processing. for two hours after field protocols and for the remaining The fresh cones are placed in a dry and well- day at a temperature of 20 °C. The germination rate and ventilated place where they are gradually opened over the germination energy are calculated after seven and 14 days. next few weeks. Opening is accelerated by processing in a ISTA protocols prescribe temperatures between 20 and conifer oven at temperatures between 27-38 °C. Drying at 30 °C (16 hours + 8 hours day). The first count is after seven 20 °C takes 20-25 days, At 40-45 °C, only two days. The days, the last on the 21st day. A germinated seed is a seed in seeds remaining in the cones are extracted by shaking which the seed root has grown to at least half the length of them in a rotating drum. If it is excreted in running water the seed. At each count, the germinated and rotten seeds on the screen, storage is not possible. are removed. Germination is expressed as the average of For cleaning, screens with apertures of 3 mm the germination of four seed samples. (for larger impurities) and 1.5 mm (for dust, etc.) are used. For planting in the forest, seedlings of the 1 But it is difficult to tell the difference between a full and + 0 form are usually used. Sowing is usually carried out an empty seed. For example, in Germany, the purity was between March and April. Use 0.5 to 1 kg of seed per 100 stated as 60% and the germination rate as 40%. m2. The cover layer is approximately 5 mm thick. Less seed The seeds have a moisture content of 8-9% when is sown in the rows and double the amount is sown over they emerge from the cones and can therefore be stored the entire bed area. This allows them to grow 12,000-35,000 for up to 2-3 years. For long-term storage, it must be dried seedlings per bed. However, if the quality of the seed is to a humidity of 5-7% by drying at a temperature of 30 °C high, it is possible to grow 20,000 seedlings by sowing for 48 hours and can be stored for five years. only 150-200 g of seed per bed. Germination in the tree Seeds with a moisture content of around is usually low (5-15%). To cultivate quality seedlings, an 10% respire intensely at room temperature, even at appropriate sowing density is needed: for 1 + 0 seedlings, temperatures between 2 and 4 °C. If the germination % a density of 60 seedlings per m2 is appropriate, while for does not drop, the germination energy of the seed drops, 2 + 0 seedlings, only 15-20 seedlings per m2 is appropriate. indicating ageing. It should therefore be dried as much Soil moisture is a very important factor for as possible (the seed is capable of drying, orthodox). sowing success rate. Sowing alder as soon as the snow Seeds with 7% humidity can be stored for several years at melts is recommended. In March and April, the optimum temperatures between -4 and -10 °C. For longer storage, soil moisture is in the top 10-30 cm. The seedlings can also drying at 3% humidity is recommended. tolerate occasional flooding. At a later point seedlings Seeds can germinate immediately in the wild thrive better in drier soils. under favourable light and temperature conditions. After Seedlings can also be grown in pots. They are drying, however, the germination is greatly reduced and sown in 7 cm diameter pots and transplanted into 8 litre can be influenced by stratifying fresh seed in a nutrient pots in May. When growing seedlings in pots, inoculation medium at temperatures between 1 and 5 °C for 180 days. with symbiotic bacteria of the genus Frankia, which fix Even better, after this stratification, the seed should be nitrogen from the air in the root nodules, is important. frozen at a temperature of -20 °C for 3 days. It is generally Alder seedlings are very susceptible to drought agreed that the secondary dormancy of alder can be when being transplanted and excavated for planting in removed or the germination of dry seed can be improved the forest, and after just a few hours survival is greatly by stratification in snow, sand or a mixture of sand and reduced. Bare roots should be immediately covered with peat at temperatures between 1 and 5 °C for 30-60 days. soil. During transport, the root system must be protected The weight of the sample taken for quality against desiccation. 87 Seed and Seedling Data for Selected Tree Species 9.7 Wild Cherry (Prunus avium L.) Wild cherry thrives singly or in groups in mixed stands from lowlands to 700 m, rarely occurring at an altitude above 1,000 m. It grows more often on carbonate soils. The bisexual flowers bloom in April and May. Pollination is with bees. The flowers are very sensitive to frost. Pollination is not always followed by fertilisation due to allele incompatibility. This leads to inter-sterile groups that need to be taken into account in the approval of seed facilities and the layout of seed plantations. The cherries ripen in June, and at higher altitudes also in July and August. There are two wild forms, the sweet cherry and the black cherry – the wild black cherry. Each fruit contains a single drupe (endocarp) with a small elongated seed covered by a seed sheath covering an endosperm of a single layer of cells thickened around the radicle and the Figure 27: Wild cherry: habitus (drawing by Teja Milavec) two outer surfaces of the cotyledons. A drupe is a woody endocarp, incorrectly named a seed. At maturity, much seeds and fruits: number of pure pits per kg 6,000-8,100 of the crop can be plucked by birds. Ripe cherries fall to (avg. 6,700); weight per 1000 pits is 125-166 g (avg. 150 g); the ground and can be spread by birds, other animals and weight of pits per 100 kg of fruit 12-18 kg. humans. The seeds can survive one or more winters under Cherries are picked at maturity, by hand from fall and snow. Germination depends on dormancy being the tree, by shaking the tree or by gathering them in a established under favourable conditions: high humidity, net under the tree at the time of fall. The cherries must aeration and a certain sequence of changes in temperature. be protected from fermentation. They are best transported Cherry trees can bear in the nursery from the in barrels of water. Small quantities can be pitted by hand 15th year onwards, and outdoors after six years. Although or with a home-made destoner. The stones must be rinsed they flower every year, the crop is irregular because it well with water. Larger quantities are purified with rotating depends on spring frosts, bad weather for bee pollination, macerators and water over sieves. Between the endocarp etc. In the early cherry, which ripens within 60 days, the and the seed there can be a bubble of air, which makes embryo is incompletely developed and does not germinate. even a full seed float on water. Therefore, the floating Late cherry forms, which ripen in 80 days, usually have a stones, which are supposed to be empty, must be broken in germination rate of around 100%. Characteristics of cherry a sample to check the quality of the seed. Figure 26: Wild cherry: flowering stages (drawing by Teja Milavec) 88 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines Cherry pits are never sown directly after Stratified seed is used for sowing in the nursery, harvesting. If it is desirable to partially remove dormancy as frost, snow or too much standing water can interrupt the by warm stratification, the seed is sown at the beginning dormancy removal process. Non-normal seed germinates of October. This also gives the seed the necessary cold 50-70% in the nursery. In Poland, 4 kg of stones are sown stratification after the warm period. While waiting for per 100 m2 bed in rows or holes and 10-13 kg per entire sowing, the seed can be partially dried or prepared for bed. This produces 13,000-25,000 seedlings per bed. Under long-term storage. cover, it is possible to grow 80-100 seedlings per m2, The cherry is a drying seed and can be dried corresponding to 160-200 germinating seeds per m2. to a moisture content of 9-10%. After rinsing with water, the seed is spread in a thin layer on the ground, stirring occasionally. After the surface has dried, the seed is left to dry at a temperature of 20 °C in a ventilated place for about 10 days. Drying can be accelerated by blowing at the same temperature. The moisture content of the whole stone is not the same as the moisture content of the seed: a moisture content of 9-11% for the stone means a seed moisture content of 6-8%. Storage in the wild is possible for 2-3 years. Dry seeds can be stored at a low temperature of -3 °C for three years or at a temperature of -10 °C for long-term storage. After the end of storage, the seed should be gradually thawed. Cherry pits have a high degree of dormancy. Only a seed that has had its dormancy completely removed is capable of normal growth. Isolated embryos, for example, grow into physiological dwarfs. Breaking of dormancy occurs in several varieties, all of them very long. Typically, the seed is stratified: Two weeks at a temperature of 25 °C, two weeks at a temperature of 3 °C, two weeks at a temperature of 25 °C, 12-16 weeks at a temperature of 3 °C (or until germination). Alternatively, it may be preceded by six weeks at 3 °C or preceded by two weeks at a temperature of 20 °C. Stratification may take place with or without medium. For seed quality analyses, a sample of 900 g is taken and a working sample of 450 g of stones. Moisture content is determined on the whole stone or separately for the stone and the seed. Viability is determined by cutting, TTC, indigo-carmine or X-radiography. The germination test requires about six months of hot-cold stratification. Figure 28: Wild cherry: fruit development (drawing by Teja Milavec) 89 Seed and Seedling Data for Selected Tree Species 9.8 Selected Conifers 9.8.1 European Silver Fir (Abies alba Mill.) The fir tree reaches the reproductive stage at 60 years of age, with full mast production every 4-6 years and partial mast production every 2-3 years. It flowers in May and ripens in September. As the cones decay on the tree, they should be picked by climbing on the trees before ripening in September. Cones are worth harvesting if at least 50% of full seeds are visible in cross-section. A litre of fresh seed usually weighs around 400 g. Fresh seed has a moisture content between 8 and 11%. There are 15-30 seeds in 1 kg of cones and 1 kg of seed in 14,000-23,000 wingless seeds. There are Figure 30: European silver fir: habitus and germination (drawing 260-290 seeds in 1 cone. Longer storage is possible at by Anja Rupar) different moisture contents and at different freezer temperatures, preferably between 8 and 10% moisture at temperatures between -10 and -15 °C in hermetically sealed containers. The fir embryo is dormant, therefore dormancy should be removed by cold stratification for 3-7 weeks before sowing. 9.8.2 European Larch Figure 29: European silver fir: development of female (left) and ( male (right) flowers (drawing by Anja Rupar) Larix decidua Mill.) European larch reaches the reproductive stage at 25-30 years, and in the outdoors at 10-15 years. Mast production occurs every 5-10 years. It flowers from March to May, and the cones ripen from September to Novem- ber of the same year. The seeds fall from September to spring. Cones are collected from standing or fallen trees from January to May. It is also possible to spread nets on snow under large areas in stands. The cones are dried in the sun and mechanically opened in drums with scrapers when they reach 15% moisture. There are 200,000-270,000 seeds in 1 kg of seed. 100 kg of cones contain 4–7 kg of wingless seeds. 1 kg of winged seeds contains 750-800 g of wingless seeds. There are 220–280 of these in 1 kg of cones. Each cone contains up to 80 seeds. Around 60% of seeds can be empty. Seeds with a moisture content between 6% and 7% can be stored for long periods at temperatures between 0 and 10 °C. Germination is slow due to the dor- mant embryo. Cold stratification for a few to six weeks is recommended. Figure 31: European larch: cone (drawing by Teja Milavec) 90 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines 9.8.3 Norway Spruce (Picea abies Karst) Spruce trees bear fruit from 30 to 50 years of age, optimally at the age of 100 years. Spruce flowers from April to June, the cones mature from October to De- cember and drop whole from October to April. They are harvested by climbing, dried in the sun and decompose when they reach 25% moisture. On average, there are 9.4 kg of winged seeds in 100 kg of cones, 550 g of wingless seeds in 1 kg of winged seeds, there are approximately 32 seeds in 1 kg of cones, and about 400 seeds in a single cone. Freshly harvested cones in October have a moisture content of approximately 30-40%. After sun-drying, the moisture content of the cones was 16% and that of the seeds 10%. Seeds with a lower % moisture retain their germination for longer. In the seed bank of the Sloveni- an Forest Genebank, seed from the 1965 harvest still had Figure 32: Spruce: cone (drawing by Teja Milavec) 70% germination in 1995. 9.8.4 Pines (Pinus spp.) Black pines bear fruit from the age of 30, Scots pi- nes 10 to 20 years earlier. The seed year is every 3-4 years for black pine, every 3-10 years for Scots pine. It flowers from May to June, with cones maturing from September to October for black pine and from November to December for Scots pine, in both cases the following year. The seeds fall from March to April in the third year. Cones are collected by climbing trees. There are 140,000 seeds per kg of cones for black pine, and 175,000 seeds per kg for Scots pine. Black pine has 2-4 kg of pure seed in 100 kg of cones, 800 g of pure seed in 1 kg of winged seeds, and up to 50 seeds in 1 kg of cones. Scots pine has 1-2 kg of pure seed in 100 kg of cones, 700 g of pure seed in 1 kg of winged seeds, and up to 160 kg of cones in 1 kg. Seeds dried at 8% moisture can be stored for longer periods. Scots pine seeds do not need stratification before sowing. Black pine seeds should be stratified for 30-60 days in a mo- ist medium at a temperature of +5 °C. Figure 33: Black pine: habitus (drawing by Klara Jager) Figure 34: Black pine: a winged and wingless seed (left), mature open cone (centre) and branch with male strobili and female immature first-year cones (right) (drawing: Klara Jager) 91 Seed and Seedling Data for Selected Tree Species 9.8.5 Douglas Fir (Pseudotsuga menziesii (Mirb.) Franco) Of the seven species of the genus Pseudotsuga trees; and in the seed plantation, once approved, collecting Carr. the most widespread is the Douglas fir (Pseudotsuga seed only if all clones have flowered. On the basis of the menziesii (Mirb.) Franco), which occurs naturally from majority of trees of one group or the other and the likely western northern Mexico (19°N, 97°N) to eastern British epigenetic effects of external conditions on the physiology Columbia (55°N, 128°N) (Stein and Owston 2008). Fossil of seeds and young, we recommend dividing Slovenia remains have been found in North America from the Early into two provenance areas for Douglas fir: provenance Tertiary onwards, and later also in Japan and Europe (ibid.). area 1 should contain the Pohorje and Alpine regions, and Because of its commercial use, it is planted outside its provenance area 2 should contain the remaining ecological current area, in Europe, Chile, New Zealand and Australia. regions (ibid.). Two varieties have been described, i.e. coastal Douglas fir can grow to more than 100 m in (var. menziesii or viridis) and continental (var. glauca), height, 4 m in diameter and live for 1300 years. It starts to which cross-breed in the interior of British Columbia. blossom at 7 to 10 (var. menziesii) or 20 years (var. glauca). It thrives at different altitudes (275-3260 m above sea The time between high mast production is between 2 and level) and in different growing conditions, which is why 11 years. The male and female cones start to sprout in its range in North America has been divided into six seed late winter or at a young age, about a year after the buds zones, which replace the regions of provenance. Clinical are conceived. Flowering takes place from March to June, genetic variability was found for several traits in coastal cones ripen from late July to early September, and seed is and continental variants of Douglas fir. Traits include released (by gravity and wind) from August to March, and survival, growth, growth form, phenology, disease and mostly in September and October (USDA 2008). pest resistance, low-temperature resistance, technological The male “cones” cover most of the crown at the characteristics and chemical structure of the wood, and proximal part of the annual branches, drooping at about 2 cm; breeding programmes were initiated 60 years ago (ibid.). they are yellow to dark red in colour. Female cones develop For both varieties, drought tolerance increases from north more distally on annual shoots, mainly in the upper part of to south and from west to east (Westergren et al. 2018). the crown. They are still erect at the time of pollen release, It was first planted in Europe in the 19th century, about 3 cm long, and vary in colour from dark green to dark with most plantations in France, the UK and Germany (http:// red. The cones on each tree are the same colour, but the www.euforgen.org/species/pseudotsuga-menziesii/). In colours of the male and female strobiles differ. Pollination southern Europe, coastal provenances from Oregon have takes place when at least half of the cone has grown from performed better, while provenances from the transition the bud scales and can take 6 to 10 days. Fertilisation occurs zone between the two varieties, sometimes referred to as about 10 weeks later (Allen and Owens 1972, in USDA 2008). var. caesia, grow best at higher altitudes (Westergren et al. Mature cones are characterised by triple bractlets visible 2018). In Slovenia, the largest concentrated areas are in the outside the cone scales. There are two seeds on each. Inner Carniola region, which also has the largest approved The number of full seeds per cone varies greatly. For var. seed stand for the production of forest reproduction glauca, about 20 to 30 per cone from the same location, material in the »selected« category. Natural rejuvenation and for var. menziesii, 4 to 54 per cone (Olson and Silen of Duglasia has been recorded in Slovenia at several 1975, in USDA 2008). The average production of pure seed locations, in the Inner Carniola, Kočevje, Novo Mesto, Celje in a stand is about 0.45 kg per tree. The abundance of the and Pohorje regions. On the basis of molecular genetic mast production can only be confirmed two months before analysis of 215 trees from seven stands and a (as yet seed dispersal. In addition to the periodicity of flowering, unapproved) seed plantation in the Novo Mesto region, the causes that affect it are poor pollination, spring frosts, it was found that the overall genetic diversity in Slovenia cone dieback, insects and other pests on the cones (at is lower than in the natural range, and the heterozygosity least nine common species in S. America) and other factors is similar, while the latter in German and Austrian stands (Owens et al. 1991, in USDA 2008). However, the potential is also mostly lower than in the natural range. Therefore, for fructification can be estimated as early as 12 months in in line with recommendations from Central Europe, we advance from the number of female buds or 17 months in recommend collecting seed from at least 20 Douglas fir advance from the number of male buds. 92 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines minimise damage from diseases and pests (e.g. rinsing with cold or hot water, fungicides, bleach, peroxide, ethanol, polyethylene glycol or ethylene (ibid.)). Stratification is mostly seed without medium after imbibition in water for 24 hours for 3-6 weeks at 2-5°C. Germination is often not equal to established viability (TTC); depending on the position of the seed at germination. Seed pre-preparation and stratification time must be determined on a lot-by-lot basis. In nursery practice, bare-root seedlings of 1 + 0, 3 + 0 and 1 + 1, 1 + 2, 2 + 1, 2 + 2 or one-year container seedlings are used. Nursery practices must take into account measures to preserve the genetic diversity of the Figure 35: Douglas fir: twig and cone (drawing by Teja Milavec) seedlings. Stratified seed is mostly sown in late winter or early spring to avoid damage over winter. After imbibition The cones start to be harvested 3-4 weeks before for 24 to 48 hours, it is refrigerated (usually in 2 kg bags) at seed set, from August onwards. Maturity is best assessed by -5°C for 28 to 60, sometimes up to 90 days. The stratified cross-sectioning the cone – the seed should be light to dark seed can be stored for later resistance at 2°C for 3 months brown, the wings light brown, the embryo should fill most or dried to a humidity of 7-15% at -7 to +3°C for 9 months of the volume and be yellow-green. The economic viability or more. of seed production is assessed by counting the number of For the production of bare-root seedlings, the full seeds – at least 5 per cross-section is recommended; seed is sown at a depth of 3-6 mm: for 2 + 0 seedlings, the to estimate the number of seeds per cone, the number density of 1-year-old seedlings is 161-323/m2, and for 1 + of seeds per longitudinal cross-section is multiplied by a 1 seedlings 538-754/m2. The recommended soil pH is 5.0- factor of 4.5 (USDA 2008). Proper storage of the cones in a 6.5. Fertilisation is mostly finished in July or late August. dry and well-ventilated place at temperatures between 7 Container seedlings can be prepared for planting within and 10 °C for 2-4 months can contribute to seed quality. one year, the recommended pH of the substrate is 4.5-6. Cones open at a loss of 35-51% of wet weight. Drying takes To improve quality, seedlings can be grown in containers place from 4 to 60 days in the air or from 2 to 48 days in in the first year and in a bed in the second year. an oven at 32-43 °C. When extracting, it must be borne in Vegetative propagation by cuttings can also be mind that higher temperatures and rough handling (robust used to increase production. They take root in the first year, extraction machines) are very damaging to the seed. The are transplanted in autumn and are ready for planting the seeds are stored at a humidity of 5-9% (on a wet weight following year. Cuttings from juvenile wood root better basis). High germination (85-87%) was still observed in and have less plagiotropic properties than cuttings from coastal Douglas fir after 25 years of storage at -18 °C, but older wood. Juvenile wood can be preserved by pruning declined within a few years at temperatures between 0 the parent tree. and 5 °C. 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Zbornik gozdarstva in lesarstva, 10: pp. 5–64. H., Bordács, S., Alizoti, P., A’Hara, S., Frank, A., Proschowsky, G. 96 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines F., Frýdl, J., Geburek, T., Guibert, M., Ivanković, M., Jurše, A., Ken- Cham, Springer International Publishing AG.: pp. 29–47. DOI: nedy, S., Kowalczyk, J., Liesebach, H., Maaten, T., Pilipović, A., 10.1007/978- 3-319-95267-3_3. Proietti, R., Schneck, V., Servais, A., Skúlason, B., Sperisen, C., • Kraigher, H., Grecs, Z., Vomer, B. and Žitnik, S. 2000. Strokov- Wolter, F., Yüksel, T. and Bozzano, M. 2021. Genetic aspects in ne usmeritve, operativna organiziranost in nadzor oskrbe z production and use of forest reproductive material: collecting gozdnim reprodukcijskim materialom. Gozdarski vestnik. 58, scientific evidence to support the development of guidelines 9: pp. 405–411. ces Programme (EUFORGEN), European Forest Institute. • Kutnar, L., Zupančič, M., Robič, D., Zupančič, N., Žitnik, S., Kralj, and decision support tools. European Forest Genetic Resour- • Grecs, Z. 1996. Obnova gozdov s saditvijo – korak k višji kako- limitation of the regions of provenance of forest tree species T., Tavčar, I., Dolinar, M., Zrnec, C. and Kraigher, H. 2002. The de- vostni ravni gozdarske operativne stroke. Zbornik gozdarstva in Slovenia based on ecological regions. Zbornik gozdarstva in in lesarstva, 51: pp. 133–143. lesarstva, 67: pp. 73–117. • Grecs, Z. 2000. Obnova gozdov s sadnjo in setvijo ter operativ- • Kutnar, L., Žitnik, S. and Kraigher, H. 2000. Razmejitev proveni- na organiziranost oskrbe z gozdnim reprodukcijskim materia- enčnih območij na osnovi fitogeografskih kriterijev. Gozdarski lom. Gozdarski vestnik, 58, 9: pp. 401–404. vestnik, 58, 9: pp. 355–360. • Grecs, Z., Kraigher, H. 2000. Gozdno semenarstvo in drevesni- • Larcher, W. 1995. Physiological plant ecology: ecophysiology čarstvo: od sestoja do sadike: zbornik. Ljubljana, Zavod za goz- and stress physiology of functional groups. Berlin, Heidelberg, dove Slovenije: Slovenian Forestry Institute. New York, Springer • Hazard, C., Kruitbos, L., Davidson, H., T. Mbow, F., Taylor, F. S. • Lipovšek, M. 1961. Pridobivanje macesnovega semena. Gozdar- A. and Johnson, D. 2017. Strain identity of the ectomycorrhi- ski vestnik, 19: pp. 95–97. • Pavle, M. 1985. Proučevanje in biološko vrednotenje semen- Horvat-Marolt, S. 1970. Stanje in razvojne tendence v gozdnem skih sestojev. Optimalna proizvodnja in predelava lesa. Lju- semenarstvu in drevesničarstvu. Ljubljana, IGLG. bljana, IGLG. • Horvat-Marolt, S. 1978. Kakovost in izbor sadik gozdnega drevja • Pavle, M. 1987. Semenski sestoji v Sloveniji: register. Ljubljana, v Sloveniji. Gozdarski vestnik, 36, 5: pp. 211–221. Inštitut za gozdno in lesno gospodarstvo. • Jerše, M. in Batič, F. 2007. Morfološka analiza puhastega hrasta • Pavle, M. 1990. Izbor in testiranje semenskih objektov. Ljublja- (Quercus pubescens Willd.) v Sloveniji. Zbornik gozdarstva in na, IGLG. lesarstva, 83: pp. 35–45. conditions. Frontiers in microbiology, 8, p. 1874. DOI: 10.3389/ Programme. 2007. Official Gazette of the Republic of Slovenia, No. 111/07. fmicb.2017.01874 • regulating plant and fungal performance under nutrient rich • NPRG. 2007. Resolution on the National Forest Development zal fungus laccaria bicolor is more important than richness in • • Pavle, M. 1990. Izbor in testiranje semenskih objektov. Ljublja- Konnert, M., Szasz-Len, A. and van Loo M. 2018 Molecular mar- na, Inštitut za gozdno in lesno gospodarstvo. kers used for genetic studies in Douglas fir (Pseudotsuga • menziesii (Mirb.) franco) In: Neophytou, C, Konnert, M. (ed.). Pavle, M. 1990. Raziskave semena in kalitve: elaborat. Ljublja- na, IGLG. Technical guidelines for molecular genetic analysis in non-na- tive forest tree species of Europe. (Studia Forestalia Slovenica, • Pavle, M. 1992. Stanje in vrednotenje semenskih sestojev 160). 1st ed. Ljubljana, Slovenian Forestry Institute, Silva Slove- gozdnega drevja v Sloveniji. Gozdarski vestnik, 50, 5-6: pp. nica Publishing Centre: pp. 6994. 277–287. • Kottke, I., Guttenberger, M., Hampp, R. in Oberwinkler F. 1987. • Pavle, M. 1993. Oblikovanje semenarskih enot na osnovi goz- An in vitro method for establishing mycorrhizae on coniferous dnih združb. Gozdarski vestnik, 51, 5/6: pp. 270–287. • Kowalski, T. 2006. Chalara fraxinea sp. nov. associated with di- • (L) Karst) germinability according to seed origin and to sto-e-back of ash (Fraxinus excelsior) in Poland. Forest Pathology, rage time in seed banks. Acta pharmaceutica, 2. suppl. 1: pp. 36, 4: pp. 264–270. DOI: 10.1111/j.1439-0329.2006.00453.x tree seedlings. Trees, 1: pp. 191–194. DOI: 10.1007/BF00193562 • Pavle, M. 1995. Determination of Norway spruce (Picea abies • Kraigher, H. 1996. Kakovostne kategorije gozdnega reproduk- • Pavle, M. 1995. Vitalnost smrekovega semena iz slovenskih se-cijskega materiala, semenske plantaže in ukrepi za izboljšanje 223–225. menskih sestojev. Gozdarski vestnik, 53: pp. 426–434. nik gozdarstva in lesarstva, No. 51). Ljubljana, Biotechnical Fa- • Pavle, M. 1997. Semenski sestoji v Sloveniji: register (Revision obroda. In: Potočnik, Igor (ed.). Kakovost v gozdarstvu: 2 (Zbor- culty, Forestry Department, Biotechnical Faculty, Department 2). Ljubljana, Inštitut za gozdno in lesno gospodarstvo. of Wood Science and Technology: Slovenian Forestry Institute: • Pavle, M., Smolej, I., Kraigher, H. and Brus, R. 1996. Noble bro- pp. 199–215. adleaves in Slovenia. In: Turok, J., Eriksson, G., Kleinschmit, J., • Canger, S. (ed.). Noble hardwoods network: report of the first Kraigher, H. 2001. Novi predpisi na področju gozdnega repro- dukcijskega materiala. In: Bogovič, M., Grecs, Z. in Kraigher, H. meeting, 24–27 March 1996, Escherode, Germany. Rome, Inter- (ed.). Gozdno semenarstvo in drevesničarstvo: strokovni se- national Plant Genetic Resources Institute: pp. 51–63. minar: program in prispevki, Kostanjevica na Krki, 11. 10. 2001. • PRGS, 1996. Program razvoja gozdov v Sloveniji. 1996. Official Ljubljana, Slovenian Forestry Institute: pp. 1–8. Gazette of the Republic of Slovenia, No. 14/1996. • Kraigher, H., Bajc, M., Božič, G., Brus, R., Jarni, K. and Westergren, • Regelungen …, 1999. Regelungen des Bundes über forstliches M. 2019. Forests, forestry and the Slovenian forest genetic re- Vermehrungsgut: Unterlagen für die Überwachungsbehörden sources programme. In: Šijačić-Nikolić, M., Milovanović, J., No- und die Kontrollbeauftragten nach dem Gesetz über forstli- nić, M. (ed.). Forests of Southeast Europe under a changing ches Saat- und Pflanzgut. Bonn, Bundesministerium für Er- climate. (Advances in Global Change Research, 65). 1st ed. nährung, Landwirtschaft und Forsten. 97 References • SO, 1971. Semenski objekti. Ljubljana, Biotehniška fakulteta v • ZGS, 1999. Poročilo Zavoda za gozdove Slovenije za leto 1998. Ljubljani, Inštitut za gozdno in lesno gospodarstvo. Ljubljana, Slovenia Forest Service. • Stein, W. I. in Owston, P. W. 2008. Pseudotsuga Carr. In: The • ZON, 1999. Nature Conservation Act. 1999. Official Gazette of woody plant seed manual. Bonner F. T. Karrfalt R. P. (ed). (Agri- the Republic of Slovenia, No. 56/99. culture Handbook, 727). Washington, DC, U. S. Department of • ZSS, 1973. Seeds and Seedlings Act. 1973. Official Gazette of the • gov/ rm/pubs_series/wo/wo_ah727.pdf • Zupančič, M. in Žagar, V. 1995. New views about the phytogeo- Westergren, M., Bajc, M. in Kraigher, H. 2018. Izvor sajenih pro- Agriculture, Forest Service: pp. 891–906. https://www.fs.usda. Socialist Republic of Slovenia, No 42/73. graphic division of Slovenia, I. Dissertationes classis IV SAZU, venienc duglazije (Pseudotsuga menziesii (Mirb.), Franco) v 36, 1: pp. 3–30. Sloveniji: poročilo. Ljubljana, Slovenian Forestry Institute. • forest in Slovenia. Forest Ecology and Management, 335: pp. Žitnik, S. 2003. Vpliv metod dodelave in shranjevanje želoda doba (Quercus robur L.) na kakovost semena in sadik: doctoral 51–59. DOI: 10.1016/j.foreco.2014.09.026 thesis. (Biotechnical Faculty, University of Ljubljana). Wraber, M. 1950a. Gojenje gozdov v luči genetike. (Strokovna in • Žitnik, S., Brus, R., Bele, J., Herman Planinšek, M., Planinšek, V., znanstvena dela, 2). Ljubljana, Inštitut za gozdarstvo in lesno Müller, C. and Kraigher, H. 2000. Praksa in razvoj v gozdnem industrijo LR Slovenije. semenarstvu in drevesničarstvu. Gozdarski vestnik, 58, 9: pp. • Wraber, M. 1950b. Fitosociologija kot temelj sodobnega gojenja 389–394. gozdov. Izvestja, 1: pp. 28–78. • • Westergren, M., Božič, G., Ferreira, A. and Kraigher, H. 2015. In- cial Gazette of the Republic of Slovenia, Nos. 24/06, 105/06, • ZUP, 2006. General Administrative Procedure Act. 2006. Offi- significant effect of management using irregular shelterwood 126/07, 65/08, 8/10, 82/13, 175/20, 3/22 http://pisrs.si/Pis.web/ system on the genetic diversity of European beech (Fagussyl- pregledPredpisa?id=ZAKO1603 vatica L.): a case study of managed stand and old growth • Žitnik, S. in Kraigher, H. 1999. Vloga fitinske kisline pri shranje- • Wraber, M. 1951. Nova pota gozdne semenarske službe. Gozdar- vanju želoda gradna (Quercus petraea (Matt.) Liebl.). Zbornik ski vestnik, 9: pp. 3–14. gozdarstva in lesarstva, 59: pp. 55–87. • ZG, 1993. Forest Act. 1993. Official Gazette of the Republic • Žitnik, S., Müller, C., Clement, A, Bonnet-Masimbert, M., Hanke, of Slovenia, Nos. 30/93, 56/99 – ZON, 67/02, 110/02 – ZGO-1, D. E. and Kraigher, H. 2000. Physiology of acorns during long-115/06 – ORZG40, 110/07, 106/10, 63/13, 101/13 – ZDavNepr, -term storage. Glasnik za šumske pokuse, 37: pp. 489–495. 17/14, 22/14 – decision of the Constitutional Court, 24/15, 9/16 – ZGGLRS and 77/16). • ZGRM, 2002. Forest Reproductive Material Act. 2002. Official Gazette of the Republic of Slovenia, Nos. 58/02, 85/02,45/04 – ZdZPKG, 77/11 10.3 Overview of Slovenian Legislation Relating to FRM • NPRG. 2007. Resolution on the National Forest Development • Rules on conditions for entry in the register of suppliers and Programme. 2007. Official Gazette of the Republic of Slove- other obligations of suppliers and on the requirements con- nia, No. 111/07. cerning the marketing of forest reproductive material (Offi- • cial Gazette of the Republic of Slovenia, Nos. 153/21, 56/22 Decree on the list of tree species and artificial hybrids. 2010. Official Gazette of the Republic of Slovenia, No. 4/10. and 92/23). • • Rules on certificates and master certificates for forest repro- Regulations on the uniform application form for plant consi- gnments, plant products and regulated articles, forest re- ductive material (Official Gazette of the Republic of Slovenia, productive material and propagating material for import Nos. 19/04, 55/12 and 155/22). inspection. 2002. Official Gazette of the Republic of Slovenia, • Rules on the demarcation of regions of provenance (Official Nos. 93/02, 93/04 and 129/20. Gazette of the Republic of Slovenia Nos. 72/03, 58/12, 69/17 • and 92/23). Mandatory Plant Health Survey of Crops and Objects, Seeds and Planting Material of Agricultural and Forest Plants. 1986. • Rules for determining data on forest seed units. 2003. Official Official Gazette of the Socialist Federal Republic of Yugosla- Gazette of the Republic of Slovenia, No. 127/03. via, Nos. 52/86, 3/87 – corr. Official Gazette of the Republic of • PRGS, 1996. Program razvoja gozdov v Sloveniji. 1996. Official • 91/03, 91/03, 91/03 and 91/03). • ZGRM, 2002. Forest Reproductive Material Act. 2002. Official Rules on the conditions to be met and the procedure for Slovenia, Nos. 111/00, 93/01, 91/03, 91/03, 91/03, 91/03, 91/03, Gazette of the Republic of Slovenia, No. 14/1996. Gazette of the Republic of Slovenia, Nos. 58/02, 85/02 – corr., of forest reproductive material of the categories “qualified” • ZON, 1999. Nature Conservation Act. 1999. Official Gazette of and “tested”. 2004. Official Gazette of the Republic of Slove-the approval of basic material intended for the production 45/04 – ZdZPKG and 77/11. nia, No. 19/04. the Republic of Slovenia, No. 56/99. • • ZSS, 1973. Seeds and Seedlings Act. 1973. Official Gazette of Rules on the requirements for the approval of basic material the Socialist Republic of Slovenia, No 42/73. in the categories “source-identified” and “selected” and on the list of approved basic material. 2003. Official Gazette of the Republic of Slovenia, No. 91/03. 98 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines OF ANNEXES TABLE 11 99 Table of Annexes ANNEX 1: Protocol for the management of forest tree seedlings from the time they are extracted from the nursery to the time they are planted, in cases where seedlings are provided by the Slovenian Forest Service from the Slovenian budget funding ANNEX 2: Documents for the approval of in situ seed facilities: • application for approval • informative document • assessment sheet ANNEX 3: Reporting form for the harvesting of forest reproductive material for the purpose of obtaining a master certificate ANNEX 4: Master Certificate of Forest Reproductive Material ANNEX 5: The certificate of the Inspectorate of Agriculture, Forestry, Hunting and Fishing (IRSKGLR) on the mixing of forest reproductive material ANNEX 6: Seed Quality Certificate issued by the Slovenian Forestry Institute 100 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines Annex 1: Protocol for the management of forest tree seedlings from the time they are extracted from the nursery to the time they are planted, in cases where seedlings are provided by the Slovenian Forest Service from the Slovenian budget funding Date: 16 September 2019 Protocol on the management of forest tree seedlings from the time of excavation in the tree nursery to the time of planting in the forest in cases where the seedlings are provided by the Slovenian Forest Service from the budget of the Republic of Slovenia 1. Purpose of the Protocol In addition to the quality of the seedlings, the success of reforestation through the planting of forest tree seed-lings depends to a large extent on how they are handled from the time they are dug up in the nursery to the time they are planted in the forest. Improper handling causes desiccation and drought stress, root rot, pest infestation, moulds and diseases, thus reducing the vitality of the seedlings. The ability of the roots to regenerate (which is even more important than the proportion of fine roots or hairs in the critical period after planting) depends mainly on the water balance of the seedlings. Forest seedlings should be exposed to dry air, strong winds or direct sunlight for as short a time as possible before planting. Desiccation can occur: ा during excavation in the nursery; ा in the nursery while waiting for transport; ा during transport; ा after transport is completed, due to late and poor backfilling; ा during planting, due to the seedlings being carried sloppily around the site. The purpose of this protocol for the management of forest tree seedlings is to ensure that the work in these phases is properly organised to ensure that regeneration by planting is successful. 3. Description of the procedures for each project stage FROM SEEDLING EXTRACTION IN THE NURSERY TO THE CUSTOMER’S ACCEPTANCE (SFS) 1 As a general rule, the sooner the seedlings are planted in the nursery after digging, the better their ST chances of survival. STAGE The nursery or supplier should keep the seedlings in a dark, cool place or cold store until transport to the worksite and ensure that they are properly cared for. During transport, the seedlings must be protected from wind and sun to avoid physiological desiccation. Transport is only possible with a protective tarpaulin or in an enclosed means of transport. 101 Table of Annexes CLIENT’S (SFS) COLLECTION OF SEEDLINGS 2 The seedlings are taken over by the purchaser (SFS) and the delivery is checked for quantity, quality, ND breeding form, origin and the adequacy of the supplier's document as defined in Article 15 of the Forest Reproductive Material Act. STAGE The quality and quantity control of the growing forms and heights shall take account of the quality requirements of the seedlings as laid down in the supply contracts concluded with the suppliers. If the seedlings delivered correspond to the order, the delivery note is validated. THE FOREST OWNER TAKES OVER THE SEEDLINGS FROM THE SFS STAGE 3RD The seedlings are taken over by the forest owner or manager, who checks that the quantity and quality by tree species are in accordance with the SFS decision. If the quantity and quality of the seedlings comply with the SFS decision, the forest owner or manager signs (acknowledges) the receipt certificate prepared by the SFS. In the case of partial deliveries of seedlings, partial certificates will be issued. 4 TRANSPORT OF SEEDLINGS TO THE WORK SITE TH STAGE During transport, the seedlings must be protected from wind and sun to avoid physiological desiccation. Transport is only possible with a protective tarpaulin or in an enclosed means of transport. STORING SEEDLINGS ON A WORKSITE IN THE FOREST Planting must be carried out immediately after delivery to the forest work site, or within 10 days at the latest. This time limit is set by a decision of the SFS. If the seedlings are stored on the site for a short period, they must be adequately protected against desiccation. Two methods are known: storage in backfill and storage under metallised, reflective foil or 5 tarpaulins. TH STAGE When storing in backfill, the bunches of seedlings should be untied and moist soil (not humus) should be spread over the roots so that they are all in contact with it. For large numbers of seedlings, the trenches have to be dug by machine. Watering should keep the soil in the backfill moist at all times, and the seedlings should not be soaked or watered by the roots. Do not store seedlings in PVC bags or under PVC foil, as the air in or under the bags becomes very hot in sunny weather, which is harmful to the seedlings and promotes the development of mould and disease. 6 PLANTING SEEDLINGS TH STAGE For planting, you will need a bag or sack to carry the seedlings; a tunnel, a pick or a planter to dig the hole (container seedlings). 102 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines The area to be planted must be well prepared. The cuttings must be removed or piled in rows so as not to interfere with planting. Where necessary, the foliage is also removed (partially or completely), shrubs and other vegetation on the area to be restored. In this task, care must be taken to create microclimatic conditions that are favourable to the further development of the seedlings. In particular, we try to preserve shrubs and trees that can mitigate against sunburn, drought, frost, etc., bearing in mind that these specimens may compete with, or damage, the seedlings planted in the future if they are removed at a later date. The seedlings must be well protected from desiccation and mechanical damage during transport around the site and must be transported in canvas bags. The best way to plant conventionally grown seedlings is in planting holes. Before digging the hole, remove the top layer of thatch, litter or grass. The planting hole should be wide enough to allow the root ball to spread out at the bottom, or deep enough to avoid damaging the taproot (e.g. in oaks). The seedling should be planted as deep as in the nursery. When excavating, we sort the soil, separating the upper humic layer from the lighter layer below. STAGE 6 When planting container-grown seedlings, the planter is used to make a hole in the soil by pushing TH it all the way in (the hoe) and rotating it at least 180° to the left and right. The use of a pick for planting is only allowed on karst terrain. Then lift the resulting stopper with your fingers. Never plant in overgrown areas. The seedlings must be planted as deep as in the containers; those planted too shallow tend to dry out. The soil around the planted seedlings is tamped down (even firm pressure with the hands or foot is enough), allowing the soil in the container to adhere to the soil in which it is planted. When marking seedlings with stakes, they are driven into the planting hole in such a way that the roots are not damaged. A humus layer of excavated soil is placed at the bottom of the planting hole and the seedling’s root ball is spread on it in its natural position. If the roots are very loose and tangled, the root ball should be loosened slightly, but care must be taken not to damage it. Then cover the roots with soil (first the darker humic soil, then the lighter soil below), gently pressing it down with your hands. There must be no empty spaces at the root. Then fill the hole all the way with the remaining soil and gently push it down with your foot to avoid damaging the roots. After planting, cover the ground around the seedling with dry leaves and grass to retain moisture and protect the roots from drying out. Responsibilities/supervision Responsibilities for each stage of the management of forest tree seedlings are: PHASES OF WORK ACCOUNTABILITY SUPERVISION 1ST STAGE supplier Forestry Inspection Service 2ND STAGE SFS Forestry Inspection Service 3RD STAGE forest owner/manager SFS 4TH STAGE forest owner/manager SFS 5TH STAGE forest owner/manager SFS 6TH STAGE forest owner/manager SFS, Forest Inspection Service 103 Table of Annexes Annex 2: Documents for the approval of in situ seed facilities: • application for approval • informative document • assessment sheet Form A: Applications for approval of a forest seed facility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . space for stamp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (name and address or name and registered office of the applicant) Gozdarski inštitut Slovenije Večna pot 2 Ljubljana SUBJECT: Application for approval of a forest seed facility I request approval of a forest seed production facility for the production of forest reproductive material in the category: ‪ source identified ‪ selected The use of harvested forest reproductive material in the “selected” category will be: ‪ multifunctional ‪ for planting in forests with limited timber production potential. In the event that the forest seed facility does not meet the conditions for approval under the category “selected”, ‪ I agree ‪ I disagree that the approval procedure is conducted under the category “source identified”. Annexes: ‪ Information on the owner of the forest seed facility and on the location of the forest seed facility ‪ Declaration of ownership ‪ Power of representation of the owner of a forest seed facility in the approval procedure Place and date...................................... Signature .................................................. 104 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines Form B: Information on the location and owner of the forest seed facility Information on the forest seed facility Municipality: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cadastral municipality: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Land plot number: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tree species name: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Information on the owner of the forest seed facility Name or company: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Street and house No.: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Place: Mail address . . . . . . . . . . . . . . . . . . . . . Postal code ‪ ‪ ‪ ‪ Municipality: . . . . . . . . . . . . . . . . . . . . . . . . Personal ID No.: /to be filled in by a natural person and sole proprietor/ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ Company reg. No.: /filled in by sole proprietor and legal person/ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ company reg. No. unit of a Tax ID No.: ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ Responsible person of the legal person: Name and surname: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Street and house No.: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Place: Mail address . . . . . . . . . . . . . . . . . . . . . Postal code Municipality: . . . . . . . . . . . . . . . . . . . . . . . Personal ID No.: /to be filled in by a natural person and sole proprietor/ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ Company reg. No.: /filled in by sole proprietor and legal person/ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ company reg. No. unit of a Tax ID No.: ‪ ‪ ‪ ‪ ‪ ‪ ‪ ‪ 105 Table of Annexes Description form To be completed by SFI Application number: Owner code: Municipality: Cadastral municipality: Land plot No.: Botanical name of the species: To be completed by SFS location: forest management unit: division, section: provenance: region of provenance: latitude: longitude: the competent local SFS unit: description occurrence: of the forest: surface area: ha age: increment: m3/ha/year timber stock: m3/ha region of provenance: 1. % 4. % 2. % 5. % 3. % 6. % economic increment: m3/ha/year growing stock m3/ha class site: community: site index: altitude: m slope % exposure: soil access: comments: compiled by: first name last name: place and date: signature: 106 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines Assessment form Application number: Owner code: Type: ‪ stand ‪ seed tree group Population size Isolation Surface area: Distance to the nearest stand of the same species, which is: Number of trees/ groups of trees: Significantly poorer quality: Distance between trees/ groups of trees: Non-indigenous/source unidentified: Uniformity Degree of variability of morphological characters: low normal high Adaptability1: Stand adapted/ not adapted to the site conditions Notes: Health condition and immunity Signs of disease/pests present: Resilience assessment: Stand productivity2: average above average below average Wood quality3: below average average above average Form and manner of growth Proportion of trees exhibiting major defects: Place and date: Present: Name and surname: Signature: Members of the commission: 1. 2. 3. Others invited: to be completed by the SFI after the decision has been issued: forest seed facility: is approved in the “selected” category and has been assigned is approved in the “source identified” category and has been not approved a number in the register of forest seed facilities: assigned a number in the register of forest seed facilities: 107 Table of Annexes Form for drawing up the list A B C D E F G H I J K L M N O P R S T Ref. No. Country Type Category ID No. Provenance Latitude Longitude Altitude Type Surface area Source Origin – in more detail Purpose Notes Ownership Municipality Cadastral municipality SFS regional unit SFS local unit 108 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines Annex 3: Reporting form for the harvesting of forest reproductive material for the purpose of obtaining a master certificate (name and address of the applicant) space for stamp (name and address of the competent unit of the Slovenian Forest Service) Pursuant to Article 14 of the Forest Reproductive Material Act (Official Gazette of the Republic of Slovenia, No 58/02 and 85/02 – corr.), is hereby notified of the procedure for obtaining forest reproductive material from the forest seed facility described below and requests the issue of a certificate of origin for the forest reproductive material: forest seed facility registration number: category: botanical name: detailed description the place of production: name and address, or company name and registered office of the supplier: the name and surname of the person responsible: the owner of the forest seed facility: estimated time of from: to: production: It is intended to produce: Seed material plants for natural regeneration plant parts The master certificate of identity of forest reproductive material is sought: seed; more precise data on the quantity of pure seed produced will be reported to the Slovenian Forestry Institute at a later stage, after the completion of the processing process on the premises; forest reproductive material produced from a forest seed facility. Place and date: Signature: Certificate of origin of forest reproductive material SFS / / SFI / Information on the quantity produced: date total hl/kg/pcs signature We certify that the forest reproductive material described above was obtained under the supervision of the Slovenia Forest Service/ Slovenian Forestry Institute in accordance with the Forest Regulations and the Regulations on Forest Reproductive Material. Date: Name and surname of the authorised person: Stamp: Signature: 109 Table of Annexes Annex 4: Master Certificate of Forest Reproductive Material (Specimen) A MASTER CERTIFICATE OF IDENTITY FOR FOREST REPRODUCTIVE MATERIAL issued in accordance with Directive 1999/105/EC SLOVENIA SI/22-010 We hereby certify that the forest reproductive material described below has been produced:  In accordance with the EC Directive; In accordance with the OECD Forest Seed and Plant Scheme. 1. Botanical name: Acer monspenssulanum L. 2. Oblika: 3. Kategorija: 4. Tip gozdnega objekta:  semenski material  znano poreklo skupina semenjakov deli rastlin izbran  sestoj sadilni material testiran 5. Purpose: 1 for multipurpose forestry 6. Reference number or statement of identity of the starting material with regard to the national register: 7.0425 . . . . . . . . . Mixture: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Source authochthonous 8. Source of the starting material (non-autochthonous if source is identified: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . non-indigenous, namely unknown 9. Country and region of provenance of the starting material: Slovenia, Submediterranean Provenance (short name): Povir - Tabor . . . . . . . . . . . 10. The altitude or altitude range of the location of the starting material: 400m/m . . . . . . . 11. Year of seed ripening/production of plants for natural regeneration: 2022 12. Quantity: 5.1kg . . . . . . . . 13. Is the lot for which this certificate is issued the result of a division of a larger lot for which an EC certificate has been issued? Yes  No The number of the previous certificate: . . . . . . . . Quantity in the previous lot: 14. Duration of tree nursery cultivation: 15. Is the planting material cultivated from seed and subsequently vegetatively propagated? Da  Ne Mode of reproduction: Number of reproductive cycles: 16. Other important information: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17. Name and address of the supplier Zavod za gozdove Slovenije CE, Večna pot 2, 1001 Ljubljana Zavod za gozdove Slovenije Hojka Kraigher CE, Večna pot 2, 1001 Ljubljana, Name and address of the supplier Stamp Name and surname of the person responsible: Slovenia Date: 5 December 2022 signature 110 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines Annex 5: The certificate of the Inspectorate of Agriculture, Forestry, Hunting and Fishing (IRSKGLR) on the mixing of forest reproductive material (name and address of the applicant) space for stamp (name and address of the competent unit of the Slovenian Forest Service) Pursuant to Articles 12 and 13 of the Forest Reproductive Material Act (Official Gazette of the Republic of Slovenia Nos. 58/02 and 85/02 – corr.), a master certificate of identity for forest reproductive material obtained by mixing is requested: region of provenance/provenance: category: botanical name: detailed description the place of production: name and address, or company name and registered office of the supplier: Certificate on the control of the mixing of forest reproductive material No. Mixture information: Master certificate No.: year of mast forest seed facility: quantity production: We hereby certify that the forest reproductive material described above was produced under the supervision of the Forestry Inspection Service, in accordance with the regulations on forest reproductive material. Date: Name and surname of the competent forestry inspector: Stamp: Signature: 111 Table of Annexes Annex 6: Seed Quality Certificate issued by the Slovenian Forestry Institute Slovenian Forestry Institute Slovenian Forestry Institute Večna pot 2, SI-1000 Ljubljana Izvid o kvaliteti semena št. (Certificate of quality of seed No.) Ime in naslov dobavitelja (Name and address of supplier): SFS Slovensko in botanično ime (English and botanical name): Registrska številka semenskega objekta (Registration number of seed facility): Številka glavnega spričevala (Number of Master Certificate): Kategorija reprodukcijskega materiala (Category of reproductive material): Namen uporabe (Usage purpose): Provencienčno območje (Region of provenance): Provenienca (Provenance): Leto v katerem so semena dozorela (Year of seed ripening): Teža partije (Lot weight): Vzorčenje opravil (Sampling done by): Število embalaž Datum vzorčenja Datum sprejema vzorca Date Datum zaključka testiranja Številka testa Number of packages Date of sampling of admission of the sample Date of conclusion of testing Number of test Analizni rezultati (Results of the analysis): velikost vzorca (size of sample): ČISTOST DELEŽ VLAGE KALIVOST GERMINATION PURITY MOISTURE CONTENT Čisto seme Weight % Uteži % Številčni % Number % % sveže teže Št. dni Normalne klice Nenormalne klice Sveže seme Trdo seme Mrtvo seme Drugo seme Inertni material fresh weight Number Normal seedlings Abnormal seedlings Fresh seeds Hard seeds Dead seeds Clean seed Other seed Inert material of days Drugo seme (Other seed): / Opis inertnega materiala (Description of inert material): / Druge analizne metode (Other methods of analysis): Uporabnost (Applicability): Vitalnost [Številčni %] Sumljivi embrii Kalivost / vitalnost Teža 1000 Mrtvi embrii Živi embrii Gluho seme [Št./1 kg semen] semen [g] Suspicious Dead Vitality[Number %] Live embryos Empty seed Germination / vitality Weight of 1000 embryos embryos [No./1 kg seed] seeds [g] TTC (Tetrazolium test) 7,9 Rentgen (X-ray) Izolirani embrio (Isolated embryos) Drugo (Other) Zdravstveno stanje (Health condition): Tip poškodbe/okužbe (Type of Poškodba/okužba z (Damage / Infection by) - Številčni delež poškodovanih/okuženih semen damage / infection) slovensko in latinsko ime (English and Latin name): (Number percentage of damaged / infected seeds): Opombe (Notes):/ Veljavnost certifikata (Validaity of the cetificate): 365 dni po dnevu izstavitve (365 days after issue) Analitik (Analyst): Jana Janša Pooblaščena strokovna oseba Responsible Officer Hojka Kraigher Datum (Date): Izvid je izdan v skladu z Zakonom o gozdnem reprodukcijskem materialu (Ur. 1. RS, št. 58/2002 in 85/2002) in Pravilnikom o ugotavljanju podatkov za seme gozdnega drevja (Ur. 1. RS, št. 127/2003) Certificate is issud in accordance with Forest Reproductive Material Act (Ur. 1. RS, št. 58/2002 in 85/2002) and Rules for determing data on forest seeds (Ur. 1. RS, št. 127/2003) 112 Conservation of Forest Genetic Resources with Forest Reproductive Material Management Guidelines HOJKA KRAIGHER Gozdarski inštitut Slovenije, Ljubljana, Slovenia hojka.kraigher@gozdis.si The textbook is aimed at students and practitioners in the field of biodiversity conservation, with an emphasis on the conservation of genetic diversity, forest breeders, forest seeders and arborists. Presentation of the basics of forest genetics, the importance and theoretical background for the conservation of forest genetic resources, the Strategy for the Conservation of Forest Genetic Resources in Europe and the EUFORGEN and SIFORGEN (Slovenian Programme for the Conservation of Forest Genetic Resources) programmes. Introduction to the botanical basics of seed structure and germi-nation physiology. Technologies for the production, processing, storage and germination of seeds of forest tree species. The basics of seedling rearing in forest nurseries. European and Slovenian legislation on forest reproductive material. Examples of documents used for the approval of forest seed facilities and certification of forest reproductive material. Keywords: genetic diversity, conservation of forest genetic resources, forest reproductive material, legislation, certification DOI https://doi.org/10.18690/um.fnm.2.2025 ISBN 978-961-286-949-6