Encapsulation of moss buds: an efficient method for the in vitro conservation and regeneration of the endangered moss <Emphasis Type="Italic">Splachnum ampullaceum</Emphasis>

In vitro culture techniques are usually employed for ex situ conservation of endangered plant species. However, encapsulation to preserve threatened bryophytes is scarcely used, and only as a pretreatment prior to cryopreservation. In our study, two different methods of germplasm conservation, involving calcium-alginate encapsulation of moss material, were assessed. The plant material used was gametophyte buds (gametophores) of Splachnum ampullaceum Hedw., a rare species of moss. Moss regeneration was evaluated at different periods of time to examine the efficacy of the technique for moss germplasm conservation. The effects of encapsulation and cold storage on developmental parameters such as protonematal colony diameter, bud length, and number of buds were also studied. The results suggest that moss encapsulation with no prior treatment may be a suitable method for germplasm conservation during long periods of time. With our method survival rates as high as 50% could be reached after 2.5 years of cold storage versus 0% of 24-h cryopreserved beads. This technique together with cold storage, avoiding freezing, may be especially important in desiccation intolerant mosses.     

Ex situ conservation of plant diversity in Romania: A synthesis of threatened and endemic taxa

Romania hosts a relatively high species diversity, including 3,829 vascular and 979 non-vascular spontaneous plant taxa. Multiple national red lists exist, with the number of taxa assessed as threatened varying greatly between them, from 548 to 1,438, and with number of taxa assigned to a given threat category also varying between the different sources. A composite list including all taxa mentioned in at least one of the selected red listings from Romania is required in order to compensate for this lack of consensus and to assess their ex situ conservation status. In this study, we synthesized data from the national red lists and counted 1,220 spontaneous vascular plant species and 201 subspecies, of which 77 are endemic and 76 subendemic for Romania. In addition, 18 non-red-listed endemics and 14 subendemics have been added, bringing the total to 1,453 threatened and (sub)endemic plant taxa, representing almost 38% of the total native vascular flora of Romania. Despite the large network of protected areas in Romania, many taxa are still being threatened with extinction in the region mainly due to anthropogenic pressure. Several ex situ conservation measures have been employed to assure a more substantial buffer against plant extinction in the wild, supported by thorough and adequate conservation strategies and multiple means to reintroduce taxa back to their natural habitats. Consequently, our second aim was to evaluate the ex situ conservation status of these threatened and (sub)endemic plants from Romania, focussing on both conventional methods (cultivation in botanic gardens, seed banking) and biotechnological approaches (in vitro tissue culture, medium-term storage and cryostorage). Of the 1,453 taxa included in our list, 642 (44.2%) are conserved by ex situ approaches. Of these, 524 are harboured in the most important botanic gardens throughout Romania, while 156 are currently held in long-term seed banks locally or in the Millennium Seed Bank of the Royal Botanic Garden, Kew (UK). Conversely, only 64 taxa from the list are preserved at the national level through in vitro cultures, and cryopreservation protocols have been developed for only 8 taxa. Overall, more than half of the threatened and (sub)endemic vascular flora from Romania remains unprotected outside the classical in situ conservation measures. For red-listed bryophytes, only 0.6% are preserved in national ex situ collections. Moreover, some aspects related to population genetic studies and the genetic stability of ex situ conserved plants are also briefly discussed, as essential prerequisites for applied biodiversity conservation programs. Finally, considering the distribution range of targeted taxa, we included a synthesis of biotechnological approaches at both national and international level. Our study presents not only a first assessment of the ex situ conservation status of national red listed flora, but also, to our knowledge, the most comprehensive and updated overview of the rare, threatened and (sub)endemic taxa from Romania. This evaluation will provide a supporting tool for national decision- and policy-making actions for biodiversity conservation, using both in situ and ex situ approaches. We also highlight the need for an updated red list for the Romanian flora that accurately follows the IUCN assessment criteria and protocols.     

Development of novel methods for the initiation of in vitro bryophyte cultures for conservation

The value of ex situ plant collections for conservation is increasingly recognised. The Royal Botanic Gardens, Kew (RGB, Kew) has been pioneering methods for the development of such collections with the establishment of the Millennium Seed Bank and a project for the ex situ conservation of threatened UK bryophytes (mosses, liverworts, hornworts). A broad range of bryophytes are currently held in a tissue culture collection at RBG, Kew on sucrose-free ¼ or ½ Murashige &; Skoog or Knops minimal medium. The efficacy of the novel sterilising agent Sodium dichloroisocyanurate (NaDCC) was tested on a range of taxa, utilising a variety of bryophytic initiation material. Concentrations of 1% (w/v) for 3 min and 0.5% (w/v) for 2 min, without the addition of detergent, were found to be successful in initiating cultures from sporophytes and leafy gametophores respectively. Initiation of cultures from wild-collected sporophytes was more successful than from wild-collected gametophores. However, pre-culturing of gametophore material was found to enhance success rate of procedures. Transferring visibly clean material away from contamination post-initiation was also determined to increase protocol success rate. The ability of the biocide Plant Preservative Mixture^TM (PPM^TM) to control fungal and bacterial contamination in bryophyte protonemal cultures was also tested, but not found to be suitable. However, results obtained indicated that contamination may confer resistance on the moss against the phytotoxic effects of the biocide. Methods developed have wider applicability for the establishment of in vitro collections of other threatened plants.     

Importance of <Emphasis Type="Italic">in vitro</Emphasis> technology to future conservation programmes worldwide

The latest IUCN statistics show that of over 12,000 plant species, 70% are threatened, 19% are critically endangered and 28 species are extinct in the wild. Target 8 of the Global Strategy for Plant Conservation (GSPC) highlights the importance of ex situ conservation of critically endangered plants. Long-term germplasm storage for species with recalcitrant seeds needs alternative measures. In vitro methods complement seed banking and other ex situ measures and are vital for long-term conservation. Conservation Biotechnology at RBG Kew is currently working on a number of rare and threatened recalcitrant species from biodiversity-rich areas of the world to develop good quality in vitro propagules for cryopreservation, recovery and restoration projects. The importance of successful in vitro propagation methods, transplantation technologies, cryopreservation and international networking for the integrated conservation of these species are discussed in detail.     

Conservation In vitro of threatened plants—Progress in the past decade

Summary In vitro techniques have found increasing use in the conservation of threatened plants in recent years and this trend is likely to continue as more species face risk of extinction. The Micropropagation Unit at Royal Botanic Gardens, Kew, UK (RBG Kew) has an extensive collection of in vitro plants including many threatened species from throughout the world. The long history of the unit and the range of plants cultured have enabled considerable expertise to be amassed in identifying the problems and developing experimental strategies for propagation and conservation of threatened plants. While a large body of knowledge is available on the in vitro culture of plants, there are limited publications relating to threatened plant conservation. This review highlights the progress in in vitro culture and conservation of threatened plants in the past decade (1995–2005) and suggests future research directions. Works on non-threatened plants are also included wherever methods have applications in rare plant conservation. Recalcitrant plant materials collected from the wild or ex situ collections are difficult to grow in culture. Different methods of sterilization and other treatments to establish clean material for culture initiation are reviewed. Application of different culture methods for multiplication, and use of unconventional materials for rooting and transplantation are reviewed. As the available plant material for culture initiation is scarce and in many cases associated with inherent problems such as low viability and endogenous contamination, reliable protocols on multiplication, rooting, and storage methods are very important. In this context, photoautotrophic micropropagation has the potential for development as a routine method for the in vitro conservation of endangered plants. Long-term storage of material in culture is challenging and the potential applications of cryopreservation are significant in this area. Future conservation biotechnology research and its applications must be aimed at conserving highly threatened, mainly endemic, plants from conservation hotspots.     

Advances in understanding the fundamental aspects required for successful cryopreservation of Australian flora

Australia is host to an amazing diversity of species, many of which require conservation efforts. In vitro culture provides a tool for not only conserving these threatened species but allows for their propagation from limited starting material. Cryopreservation provides the greatest long-term storage option for in vitro cultures and as a conservation tool for other germplasm. However, while cryopreservation has proven capable of delivering viable long-term storage with some plant taxa, the process of deriving protocols is still largely an incremental process. The key to faster and more intuitive optimising of cryopreservation protocols lies with continuing to develop a better understanding of key factors, including issues with plant physiology (such as genetic stability, the composition of the proteome and metabolome, cell membrane characteristics, and antioxidant defences) and how the stresses imposed by cryopreservation (such as the excision damage, desiccation, cryoprotective agent toxicity, ice crystal damage, and cooling to cryogenic temperatures) interact and contribute to the cryocapability of a species. This review focuses on the advances that have been made towards understanding cryogenic stress and how this has led to improved cryopreservation protocols, in the context of cryopreserving Australian flora.     

种质资源保存的战略问题和面临的挑战

植物种质资源保存,特别是种子库保存是各种迁地保护措施中最为经济有效的方法。通过对成千上万个物种的有效保存,种子库为减缓物种的灭绝和气候变化对生物多样性的影响发挥了特别关键的作用。本文较为详细地介绍了“中国西南野生生物种质资源库”的立项背景和最新进展,同时介绍了世界上其它几个主要的植物迁地保存设施,特别是英国皇家植物园的“千年种子库”。结合“全球植物保护策略”讨论了中国植物濒危状况,估计我国受威胁的物种比例达20—25％,甚至更高。本文还简要讨论了种子保存中的一些科学问题,包括超低温保存,并强调了植物分类学和种子生物学的学科建设在植物种质资源保护中的重要意义。     

Germination capacity and viability of threatened species collections in seed banks

Facing the current biodiversity crisis, the value of ex situ conservation has been increasingly acknowledged in international treaties and legislations. Seed banks are a good way of conserving biodiversity, providing that seeds are of high quality and at maximum viability. However, despite the number of established ex situ facilities, there is little information on seed viability in botanic garden seed banks. This paper analyses the status of the seed collection of the National Botanic Garden of Belgium by determining the germination capacity and viability of seeds that have been stored for 1–26 years. It aims at: (1) ensuring that existing storage conditions provide effective ex situ conservation of threatened species; (2) providing viability data on threatened species; (3) planning future collection and storage efforts for seeds of West European species of conservation value. Results from this study showed that the germination and viability percentages of the 250 tested species reached on average 59 and 79% respectively. Some families typically performed better than others. Within a species, consistent results were not always obtained. Over a quarter of accessions exhibited some degree of dormancy. Considering the current lack of knowledge in seed germination and dormancy of many rare and threatened species, we believe that the quality of a seed collection should be estimated by its viability and not by its germination percentage. This study calls for further research in order to better understand the biology of a range of threatened native species.     

Cryopreservation of the endangered mahseer (Tor khudree) spermatozoa: I. Effect of extender composition, cryoprotectants, dilution ratio, and storage period on post-thaw viability

Several in situ and ex situ conservation strategies have been suggested for the revival of stocks of Tor khudree (Sykes), a threatened species. Cryopreservation of spermatozoa is crucial for the conservation of stocks of endangered species so that sustainable production can be ensured. Among the different extenders, modified fish Ringer (E1) was found to be the best for cryopreservation of T. khudree spermatozoa. Extender E2 appeared the next best. Extenders based on chicken egg yolk and milk powder were found to be unsuitable for the cryopreservation of T. khudree spermatozoa. Among the cryoprotectants, dimethyl sulfoxide provided maximum protection to spermatozoa during freezing and thawing. Propylene glycol and methanol were found to be less effective. Of the four spermatozoa dilutions, 1:10, 1:15, and 1:20 showed better motility rates than 1:5. At the former dilution ratios, the motility rates which were more than 95% prior to freezing were reduced to 80-81 and 43-67%, 10 and 70 days after cryopreservation, respectively. The motility duration did not differ much with increasing storage period at all the dilution ratios. Motility rates generally decreased with an increase in frozen storage. When spermatozoa were thawed and stored at 25 degrees C for varying periods, motility percentage, and duration decreased gradually as the storage period increased; spermatozoa stored up to 40 min after thawing retained 55% motility and were motile up to 77s; these values declined further leading to the complete cessation of motility 70 min after storage. The importance of extender-cryoprotectant mixture, milt dilution, and storage period in developing a protocol for T. khudree spermatozoa cryopreservation is discussed.     

<Emphasis Type="Italic">In vitro</Emphasis> conservation of European bryophytes

The use of in vitro techniques for conservation has been rising steadily since their inclusion in The Convention on Biological Diversity and The Global Strategy for Plant Conservation. Unfortunately, bryophytes are often overlooked in conservation initiatives, but they are important in a number of large-scale ecosystem processes, i.e. nutrient, water and carbon cycling. There is a long history of the use of tissue culture in cultivating bryophytes, and many species respond well to in vitro techniques. For 6 yr (2000–2006), The Royal Botanic Gardens, Kew and the UK statutory conservation agencies supported a project for the ex situ conservation of bryophytes. Living and cryopreserved collections of UK threatened species were successfully established and the cryopreserved collection continues to be maintained. Other in vitro conservation collections are maintained over Europe, at botanic gardens, museums and by individual university researchers, but there is no coherent European collection of bryophytes for conservation, or standardisation of techniques. A major issue for many in vitro collections is the maintenance of within species genetic diversity. Such diversity is considered to be important, as it is the basis by which populations of species can adapt to new conditions and evolve. We are proposing to establish a European network for in vitro conservation of bryophytes. We envisage that this will include living collections, cryopreserved collections and spore collections. Conservation of genetic diversity would be a priority and the collections would provide a valuable resource for conservation initiatives and support research into rare and threatened species.     

Development and large scale application of cryopreservation techniques for shoot and somatic embryo cultures of tropical crops

Shoot-tips and somatic embryos are the explants of choice for the in vitro long-term storage of ex situ plant genetic resources in liquid nitrogen. Cryopreservation of organized structures has significantly progressed, especially for species of tropical origin, with the development of several vitrification-based procedures such as encapsulation-dehydration, vitrification and droplet-vitrification approaches. They have allowed improvements in survival and recovery after cryopreservation compared with conventional crystallization-based protocols, proving their effectiveness for large scale application with embryos and shoot-tips of different plants. This review addresses the main physical and technological aspects involved in plant cryopreservation methods, illustrating the development of research with three cases: citrus, cassava and potato. These studies demonstrate how cryopreservation strategies are increasingly applied for their successful employment in the genebanks.     

Use of biotechnologies for the conservation of plant biodiversity

In vitro techniques are very useful for conserving plant biodiversity, including (a) genetic resources of recalcitrant seed and vegetatively propagated species, (b) rare and endangered plant species and (c) biotechnology products such as elite genotypes and genetically engineered material. Explants from recalcitrant seed and vegetatively propagated species can be efficiently collected under field conditions using in vitro techniques. In vitro culture techniques ensure the production and rapid multiplication of disease-free material. Medium-term conservation is achieved by reducing growth of plant material, thus increasing intervals between subcultures. For long-term conservation, cryopreservation (liquid nitrogen, −196°C) allows storing plant material without modification or alteration for extended periods, protected from contaminations and with limited maintenance. Slow growth storage protocols are routinely employed for a large number of species, including numerous endangered plants, from temperate and tropical origin. Cryopreservation is well advanced for vegetatively propagated species, and techniques are ready for large-scale experimentation in an increasing number of cases. Research is much less advanced for recalcitrant species due to their seed characteristics, viz., very high sensitivity to desiccation, structural complexity and heterogeneity in terms of developmental stage and water content at maturity. However, various technical approaches should be explored to develop cryopreservation techniques for a larger number of recalcitrant seed species. A range of analytical techniques are available, which allow understanding physical and biological processes taking place in explants during cryopreservation. These techniques are extremely useful to assist in the development of cryopreservation protocols. In comparison with crop species, only limited research has been performed on cryopreservation of rare and endangered species. Even though routine use of cryopreservation is still limited, an increasing number of examples where cryopreservation is used on a large scale can be found both in genebanks for crops and in botanical gardens for endangered species.     

Cryopreservation of Cymbidium eburneum Lindl. and C. hookerianum Rchb. f., two threatened and vulnerable orchids via encapsulation–dehydration

A successful cryopreservation protocol for the long-term conservation of protocorms of two threatened and vulnerable orchids, Cymbidium eburneum Lindl. and Cymbidium hookerianum Rchb. f., was developed using encapsulation–dehydration. Protocorms were osmoprotected in liquid Murashige and Skoog medium (MS) containing 0.7 M sucrose for 20 h at 25?±?2°C on a rotary shaker, and incorporated into an encapsulation matrix [consisting of 3% (w/v) sodium alginate and 100 mM CaCl₂]. The encapsulated protocorms, which were desiccated in a laminar airflow cabinet for 6 h, were able to withstand cryostorage in liquid nitrogen. Maximum regeneration into complete plantlets (72% for C. eburneum and 70% for C. hookerianum) of the cryostored, encapsulated protocorms was obtained using MS medium containing 3% sucrose and 0.8% agar. Using this protocol of cryopreservation, long-term preservation for ex situ conservation of these two threatened orchids can be accomplished.     

Plant cryopreservation: Progress and prospects

Summary Cryopreservation (liquid nitrogen, −196°C) represents the only safe and cost-effective option for long-term conservation of germplasm of non-orthodox seed species, vegetatively propagated species, and of biotechnology products. Classical cryopreservation techniques, which are based on freeze-induced dehydration, are mainly employed for freezing undifferentiated cultures and apices of cold-tolerant species. New cryopreservation techniques, which are based on vitrification of internal solutes, are successfully employed with all explant types, including cells suspensions and calluses, apices, and somatic and zygotic embryos of temperate and tropical species. The development of cryopreservation protocols is significantly more advanced for vegetatively propagated species than for recalcitrant seed species. Even though its routine use is still limited, there are a growing number of examples where cryopreservation is employed on a large scale for different types of materials, including seeds with orthodox and intermediate storage behaviour, dormant buds, pollen, biotechnology products, and apices sampled from in vitro plantlets of vegetatively propagated species. Cryopreservation can also be employed for uses other than germplasm conservation, such as cryoselection, i.e., the selection through freezing of samples with special properties, or cryotherapy, i.e., the elimination of viruses from infected plants through apex cryopreservation. Because of its high potential, it is expected that cryopreservation will become more frequently employed for long-term conservation of plant genetic resources.     

Plant species with extremely small populations (PSESP) in China: A seed and spore biology perspective

Approximately one fifth of the world's plants are at risk of extinction. Of these, a significant number exist as populations of few individuals, with limited distribution ranges and under enormous pressure due to habitat destruction. In China, these most-at-risk species are described as ‘plant species with extremely small populations’ (PSESP). Implementing conservation action for such listed species is urgent. Storing seeds is one of the main means of ex situ conservation for flowering plants. Spore storage could provide a simple and economical method for fern ex situ conservation. Seed and spore germination in nature is a critical step in species regeneration and thus in situ conservation. But what is known about the seed and spore biology (storage and germination) of at-risk species? We have used China's PSESP (the first group listing) as a case study to understand the gaps in knowledge on propagule biology of threatened plant species. We found that whilst germination information is available for 28 species (23% of PSESP), storage characteristics are only known for 8% of PSESP (10 species). Moreover, we estimate that 60% of the listed species may require cryopreservation for long-term storage. We conclude that comparative biology studies are urgently needed on the world's most threatened taxa so that conservation action can progress beyond species listing.     

Mulberry biodiversity conservation through cryopreservation

A 238 mulberry germplasm accession collection from diverse regions maintained under tropical conditions was identified from an ex situ field gene bank. The purpose was to prioritize the in vitro conservation and cryopreservation to develop long-term biodiversity conservation for ensuring sustainable utilization of these valuable resources. Reliable cryo techniques using desiccation and slow freezing of winter-dormant buds were used. Storage potential of bud grafts of different Morus species at −1.5°C for 90 d indicated species-specific variation, and most of the wild species were found sensitive. In vitro regeneration and cryopreservation (−196°C) protocols using differentiated bud meristems, like axillary winter-dormant buds, were worked out for a wide range of landraces, wild, and cultivated varieties of Morus. Buds maintained under subtropical location are also amenable for cryopreservation. Successful cryopreservation of winter-dormant buds belonging to Morus indica, Morus alba, Morus latifolia, Morus cathayana, Morus laevigata, Morus nigra, Morus australis, Morus bombycis, Morus sinensis, Morus multicaulis, and Morus rotundiloba was achieved. Among wild species, Morus tiliaefolia and Morus serrata showed moderate recovery after cryopreservation. Survival rates did not alter after 3 yr of cryopreservation. Inter-simple sequence repeat markers were used to ascertain the genetic stability of cryopreserved mulberry germplasm accessions, which showed no difference detected among the plantlets regenerated from frozen apices in comparison to the nonfrozen material.     

<Emphasis Type="Italic">In Vitro</Emphasis> Preservation of Spanish Biodiversity

Spanish territories contain many of the hot spots of plant biodiversity among European countries. Most of the Spanish territory is found in the Mediterranean basin and in the Canary Islands, a region of great floral singularity and diversity (Macaronesian bioregion). Therefore, an important effort must be made to contribute to its conservation. Several strategies can be considered, but seed conservation under standard conditions is the most resource-efficient method. However, the application of this methodology is not always possible for recalcitrant seeds or species for which vegetative propagation is necessary or convenient under some circumstances. Micropropagation is one of the measures suggested for preserving endangered species. During the 1990s, several in vitro culture protocols for Spanish endemics were established. The main purpose of this strategy was to obtain a considerable number of individuals to reduce the loss of natural populations. Likewise, diverse slow growth protocols were developed for this material. However, these efforts usually did not lead to the establishment of in vitro collections. The advantages and disadvantages of the in vitro conservation strategy will be reviewed for some cases. The establishment of the in vitro protocols together with the development of cryopreservation techniques created the ideal conditions to generate cryogenic collections. In this paper, we review the knowledge and experience accumulated during the last decades in micropropagation, slow growth, and cryopreservation for Spanish plant wild species. Their application in the development ex situ collections and their contribution toward an integrated system to conserve threatened species will be discussed.     

Gaps in seed banking are compromising the GSPC’s Target 8 in a megadiverse country

Ex situ seed conservation is an effective strategy to help safeguarding plants from extinction. The updated Global Strategy for Plant Conservation’s (GSPC) Target 8 aims to include 75% of threatened plant species in ex situ collections by 2020, preferably in the country of origin. Halfway through the updated GSPC program, we evaluate the current state of knowledge and practice of ex situ seed conservation of threatened species from megadiverse Brazilian flora, contributing to this Target. We identify knowledge gaps and costs to achieve Target 8 through seed banking in Brazil within the time constraints of the GSPC and in a scenario of recent science budget funding cuts. Knowledge on seed storage behavior is available only for 24 Brazilian species (1.3%). Seed desiccation tolerance was concluded for 175 of 228 species, feasibly allowing safe storage of most Brazilian species at sub-zero temperatures. However, only 26 species (1.3%) are effectively banked in research institutions. Surprisingly, the percentage of banked threatened species hardly increased in the first 5 years since the update of the Target (0.55%, 2011–2015), and Brazil now faces the challenge of banking almost 1500 species during 2016–2020. Despite a major lack of commitment of Brazilian institutions and of knowledge to achieve the Target, the costs for banking the remaining species were estimated to be only US$3.9 million. We call for a nationwide coordinated effort of government agencies, policy makers and research institutions to include ex situ seed conservation in the environmental agenda to pursue achievement of the Target by 2020.     

Survival and genetic stability of shoot tips of <Emphasis Type="Italic">Hedeoma todsenii</Emphasis> R.S.Irving after long-term cryostorage

Endangered and rare species for which seed banking is not possible require alternative methods of ex situ conservation for long-term preservation. These methods depend primarily on cryopreservation methods, such as shoot tip cryopreservation, but there are few datasets with information on the long-term survival of shoot tips stored in liquid nitrogen. In this study, survival and genetic stability of shoot tips of the endangered species, Hedeoma todsenii, banked over multiple years were examined. In vitro cultures cryopreserved with both the encapsulation dehydration and the encapsulation vitrification methods showed good average survival after up to 13 yr of storage in liquid nitrogen. The application of droplet vitrification to this species increased survival significantly, with an average of 72%, compared with 24–45% survival obtained with other methods. As measured with microsatellite and sequence-related amplified polymorphism (SRAP) markers, the genetic stability of the same genotypes stored over different periods of time typically did not change. However, there was an average of 10.4% band loss between replicate samples that did indicate a potential change in DNA composition. These results demonstrate the use of shoot tip cryopreservation as an effective ex situ conservation tool for this species, but genetic stability of the cryopreserved tissues should be closely monitored.     

Applying red lists to the evaluation of agricultural habitat: regular occurrence of threatened birds,vascular plants,and bryophytes in field margins of Poland

Using the assessments of conservation status of flora and fauna, we discuss declining species in semi-natural field margins and stress the importance of these vanishing habitats. Seventy field margins in the diverse farmland of SW Poland were investigated with regard to their vascular plants, bryophytes and breeding birds. We checked the occurrence of threatened and conservation concern species, i.e. those listed in local, national or European threatened species lists, and birds with an unfavorable conservation status in Europe. Of a total of 673 species, 18 classified as threatened were recorded: 12 vascular plants (2.2 % of the total number of species), five bryophytes (5.6 %), and one bird species (2.0 %). Threatened plants occurred in 18.6 % of study plots, bryophytes in 20.0 % and birds in 12.9 %. Eleven bird species, noted in 95.7 % of study plots, had an unfavorable conservation status in Europe. Increasing volumes of trees and shrubs significantly increased the overall richness in each taxa. In percentage terms the occurrence of focal species in all three taxa tended to be higher in shrubby than in herbaceous and tree-lined margins. Our data demonstrate that field margins in Central European arable farmland regularly support rare and threatened species, and therefore deserve greater conservation efforts. Red lists along with alternative listing approaches can be employed to evaluate the biodiversity of fine-scale habitats, but their applicability depends on the taxonomic group and geographical scale of the lists, reflecting different conservation priorities.