The contribution of the Millennium Seed Bank Project to <Emphasis Type="Italic">ex situ</Emphasis> plant conservation in Chile

For a long time in situ conservation has been the main approach used to protect Chilean plant diversity. However, due to the high level of endemism of its flora (50%) and an increasing human impact on wild areas, ex situ conservation has become an urgent requirement to avoid the extinction of plant populations and species. Since 2001, the Instituto de Investigaciones Agropecuarias (INIA), Chile, has been working in partnership with the Royal Botanic Gardens Kew, (Kew) through the Millennium Seed Bank Project (MSB) with the objective of conserving 20% of the Chilean flora as seeds in long-term storage. This seed conservation effort has focussed mainly on the endangered and endemic plants of the Chilean drylands. Towards the end of the first phase of the MSB some 1482 seed collections representing 850 species and subspecies have been collected and safely preserved in the INIA Seed Base Bank and duplicated at Kew. Almost 70% of the total species collected are endemic to Chile and several of them are endangered. Additionally, seed germination research has been conducted for nearly 400 species and seed collections have been used to propagate several threatened species. Germination protocols have been published and disseminated online. Over 4,500 herbarium vouchers have been collected, largely duplicated at Kew and at the national herbarium in Chile. As a result of the inputs of INIA and the MSB, collaboration has been extended to other national stakeholders, mainly for plant taxonomy and seed collecting. In this context two training courses have been run for 70 staff/students. This training has contributed to the raising of general awareness of the need for the long-term protection of Chilean plant diversity and to demonstrate the key role that ex situ seed conservation can play in meeting this need.     

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.     

Strategies for agrobiodiversity conservation and promotion: a case from Yunnan, China

This paper deals with strategies for agrobiodiversity conservation and promotion based on studies on four plant groups (selected from 50 plant groups) occurring in the Yunnan Province of China. These plants are edible konjac (Amorphophallus), medicinal Paris, Musella lasiocarpa and wild tea (Camellia sinensis and its wild relatives), including their cultivars and wild populations. After analyzing the target plants, we conclude that the following strategies should be adopted to conserve and promote agrobiodiversity: (1) in situ conservation of agrobiodiversity, including habitat protection of wild populations, maintenance of native species and varieties in traditional agroecosystems, and relevant environmental education; (2) ex situ conservation and promotion of agrobiodiversity, including establishment of living collections and germplasm banks, and introduction of species and varieties into agroecosystems for agricultural practice; and (3) promotion and conservation of agrobiodiversity through sustainable uses, including technique development of propagation, cultivation, pest and disease control, on farm and off farm management, and other activities such as new variety breeding and scientific studies. Strategies developed here will be helpful to conserve and promote agrobiodiversity at agroecosystem, species, variety or landrace, and management system levels.     

Ex Situ Conservation of Orchids in a Warming World

Whilst there is overwhelming scientific evidence that dramatic changes in regional climates are likely to occur throughout the 21st century, the scientific community remains uncertain how the effects of global heating will combine with other environmental factors to affect wild orchid populations. It is, however, likely that many populations will be affected adversely and that in situ conservation techniques by themselves will not be sufficient to prevent the extinction of many species. A range of complimentary ex situ strategies are discussed. Amongst these orchid seed banking has been shown to be an invaluable tool for conserving the maximum amount of genetic diversity in the minimum space and has the potential to enable the conservation of valuable material for possible re-introduction and habitat restoration programmes in the future. The Darwin Initiative project, ‘Orchid Seed Stores for Sustainable Use’ (OSSSU), is currently establishing a global network of orchid seed banks focussing initially on countries with high orchid biodiversity in Asia and Latin America. Particular reference is made to ex situ conservation in China, together with the urgent need to gather more data to determine which habitats and species are most at risk of extinction in the wild in the immediate future.     

Biotechnology for saving rare and threatened flora in a biodiversity hotspot

The Southwest Australian Floristic Region (SWAFR) is a plant biodiversity hotspot with a geographically isolated and predominantly endemic flora. Known threatening processes (i.e. excessive clearing of native vegetation, soil salinity, soil erosion and chronic weed infestation) combined with uncertain but potentially deleterious environmental (climate) changes pose great challenges for conservation and restoration efforts. With a paucity of nature reserves, in situ protection of species can be problematic. For many species, ex situ conservation becomes the only viable strategy for saving species from extinction via seed banking or living collections established through vegetative propagation, or tissue (in vitro) culture methods. Development of specific in vitro protocols is necessary to successfully initiate culture lines, with considerable development of nutrient media, plant growth regulator regimes and incubation conditions required to optimise shoot regeneration and multiplication, especially with woody species of the SWAFR. In addition, integration of root induction and acclimatization stages has allowed significant improvements in speed and success of plant production of both endangered and difficult-to-propagate woody species. We contend that there is also considerable potential for expansion of alternative in vitro technologies such as somatic embryogenesis for difficult taxa to complement existing ex situ conservation and restoration strategies in biodiversity hotspots such as SWAFR.     

Can Biospecimen Science Expedite the Ex Situ Conservation of Plants in Megadiverse Countries? A Focus on the Flora of Brazil

Increasing the number of species conserved ex situ in Megadiverse countries is a major task exacerbated by many intricate factors including: biome complexity, wide range of biodiversity and an incomplete knowledge of life cycles, reproductive strategies, adaptations and species interactions. Although, establishing safe reserves is a crucial conservation measure their security and effective maintenance can be unfavourably compromised by climate change and the risks incurred by socioeconomic instability and changes in land use. Anthropogenic impacts, non-sustainable practices and habitat erosion have motivated current international efforts which focused on Brazil as host of ‘Rio+20’ the United Nation's twentieth anniversary conference on sustainable development. The revised targets of the Global Strategy for Plant Conservation (GSPC) are responses to species decline and realizing Target 8, which concerns ex situ conservation, places the heaviest burdens on countries that are custodians of the highest levels of global biodiversity. At the scientific level, ex situ conservation of endemic species in genebanks is often hindered by a lack of information about molecular genetics and problematic (recalcitrant) storage behaviors that restrict the preservation of flora native to Megadiverse countries. The potential for applying the ‘Biospecimen Science’ paradigm in expediting conservation in biodiversity-rich biomes is considered using Brazil as an exemplar of a Megadiverse country. The impacts of process chains on the quality of preserved plant germplasm and using evidence-based research to improve conservation outcomes, risk and quality management systems are appraised. The Biospecimen Science approach is not intended to displace conventional conservation practices but rather, to enhance their effectiveness in terms of the scale and efficiency of their scientific and technical operations.     

Is It Still Necessary to Continue to Collect Crop Genetic Resources in the Mediterranean Area? A Case Study in Catalonia

Crop genetic resources have been extensively collected in Europe in the last century, creating large, publicly available ex situ collections. While this huge genetic diversity is often underutilized, in recent decades, several initiatives have emerged at the local level to collect germplasm cultivated on farm. Uncoordinated actors often carry out these collecting missions without considering previously collected data. To explore whether new collecting missions are likely to be worthwhile, we studied the crop genetic resources conservation network in Catalonia by analyzing the passport data and geographical distribution of germplasm stored in seed banks. Moreover, to determine whether this germplasm was representative of the diversity cultivated on farm, we performed new collecting missions in four randomly selected areas in the European Union’s Natura 2000 network and compared the results with the ex situ databases. Seed banks currently hold a large germplasm collection (2931 accessions), although most materials are conserved in private collections without regulated systems for seed regeneration and are not present as duplicates in the National Inventory. One important shortcoming of the ex situ network is that the germplasm conserved ex situ shows a low geographical coverage, representing only 35.3% of the municipalities in Catalonia. Our new missions allowed us to collect 234 accessions, mostly tomatoes (17.5%) and beans (16.2%). The ecological indicators’ richness (both at species (S) and variety (V) levels), total abundance (A), and the Shannon-Weaver diversity index calculated at species (H2, considering the different accessions of each variety as a single population) and variety levels (H3, considering the intra-varietal genetic diversity) were higher in the newly collected germplasm than in the ex situ collections, suggesting that seed banks do not accurately represent the genetic diversity still cultivated on farm. Moreover, some important landraces from each area were absent or underrepresented in the ex situ collections. Thus, it is necessary to continue to devote efforts to collecting germplasm; better organization between actors and targeting specific species/varieties can increase the efficiency of new collecting missions. As a conclusion, we propose different criteria to guide new missions and to improve the network’s conservation activities.     

Criteria for assessing Italian <Emphasis Type="Italic">ex situ</Emphasis> collections of threatened plants

The ex situ conservation of plant species is a challenge for all signatories of the Convention on Biological Diversity to meet. In Italy, all conservation-related issues are supervised by the Ministry for the Environment and Protection of Land and Sea, but so far no national plan has been drafted, let alone funded, for ex situ plant conservation. As a contribution towards the establishment of a scientifically based policy, a national project has been launched, under the coordination of the Botanic Garden of Palermo. In its framework, the Botanic Garden of Pisa is developing a set of criteria for assessing Italian ex situ collections of threatened plant species. The criteria will be applied to datasets obtained by the Italian seedbanks through questionnaires. Requested data include resources and procedures, documentation systems, facilities and accessions. The expected results consist of the production of printed or electronic material concerning a) the technical-scientific profile of the Italian seed banks and of their collections; b) the ecogeographic profile of seed collections of selected species and c) the evaluation of the method used for gathering and analysing data. The project is currently under way and the results are due by September 2010. The documents and protocols which will be produced within the project will enable a stronger interaction of the Italian seed banks with other national and European seed bank networks.     

Planning for optimal conservation of geographical genetic variability within species

Systematic Conservation Planning (SCP) involves a series of steps that should be accomplished to determine the most cost-effective way to invest in conservation action. Although SCP has been usually applied at the species level (or hierarchically higher), it is possible to use alleles from molecular analyses at the population level as basic units for analyses. Here we demonstrate how SCP procedures can be used to establish optimum strategies for in situ and ex situ conservation of a single species, using Dipteryx alata (a Fabaceae tree species widely distributed and endemics to Brazilian Cerrado) as a case study. Data for the analyses consisted in 52 alleles from eight microsatellite loci coded for a total of 644 individual trees sampled in 25 local populations throughout species’ geographic range. We found optimal solutions in which seven local populations are the smallest set of local populations of D. alata that should be conserved to represent the known genetic diversity. Combining these several solutions allowed estimating the relative importance of the local populations for conserving all known alleles, taking into account the current land-use patterns in the region. A germplasm collection for this species already exists, so we also used SCP approach to identify the smallest number of populations that should be further collected in the field to complement the existing collection, showing that only four local populations should be sampled for optimizing the species ex situ representation. The initial application of the SCP methods to genetic data showed here can be a useful starting point for methodological and conceptual improvements and may be a first important step towards a comprehensive and balanced quantitative definition of conservation goals, shedding light to new possibilities for in situ and ex situ designs within species.     

Ex Situ Conservation Priorities for the Wild Relatives of Potato (Solanum L. Section Petota)

Crop wild relatives have a long history of use in potato breeding, particularly for pest and disease resistance, and are expected to be increasingly used in the search for tolerance to biotic and abiotic stresses. Their current and future use in crop improvement depends on their availability in ex situ germplasm collections. As these plants are impacted in the wild by habitat destruction and climate change, actions to ensure their conservation ex situ become ever more urgent. We analyzed the state of ex situ conservation of 73 of the closest wild relatives of potato (Solanum section Petota) with the aim of establishing priorities for further collecting to fill important gaps in germplasm collections. A total of 32 species (43.8%), were assigned high priority for further collecting due to severe gaps in their ex situ collections. Such gaps are most pronounced in the geographic center of diversity of the wild relatives in Peru. A total of 20 and 18 species were assessed as medium and low priority for further collecting, respectively, with only three species determined to be sufficiently represented currently. Priorities for further collecting include: (i) species completely lacking representation in germplasm collections; (ii) other high priority taxa, with geographic emphasis on the center of species diversity; (iii) medium priority species. Such collecting efforts combined with further emphasis on improving ex situ conservation technologies and methods, performing genotypic and phenotypic characterization of wild relative diversity, monitoring wild populations in situ, and making conserved wild relatives and their associated data accessible to the global research community, represent key steps in ensuring the long-term availability of the wild genetic resources of this important crop.     

Ex situ conservation of plant germplasm using biotechnology

Conservation of plant genetic resources attracts more and more public interest as the only way to guarantee adequate food supplies for future human generations. However, the conservation and subsequent use of such resources are complicated by cultural, economical, technical and political issues. Over the last 30 years, there have been significant increases in the number of plant collections and in accessions in ex situ storage centres throughout the World. The present review is of these ex situ collections and the contribution biotechnology has made and can make to conservation of plant germplasm. The applications and limitations of the new, molecular approaches to germplasm characterization are discussed. In vitro slow growth is used routinely for conserving germplasm of plants such as banana, plantain, cassava and potato. More recently, cryopreservation procedures have become more accessible for long-term storage. New cryopreservation techniques, such as encapsulation-dehydration, vitrification and desiccation, lengthen the list of plant species that can not only tolerate low temperatures but also give normal growth on recovery. Extensive research is still needed if these techniques are to be fully exploited.V.M. Villalobos is with the Food and Agriculture Organization of the United Nations, Viale delle Terme di Caracalia, 00100 Rome, Italy. F. Engelmann is with the International Plant Genetic Resources Institute (IPGRI), Via delle Sette Chiese 142, 00145 Rome, Italy.     

Current status and conservation of Pistacia germplasm

The genetic erosion of Pistacia germplasm has been highlighted in many reports. In order to emphasize this and to focus more attention on this subject, national and international (especially IPGRI and IFAR) institutions have initiated projects proposing to characterize, collect and conserve Pistacia germplasm. Therefore, this paper reviews recent research concerning conventional (in situ and ex situ) and unconventional biotechnological conservation strategies applied to the preservation of Pistacia germplasm. As regards conventional conservation, the majority of germplasm collections of Pistacia species are preserved on farms (in situ) and in seed and field genebanks (ex situ), as well as in the wild, where they are vulnerable to unexpected weather conditions and/or diseases. Hence, complementary successful unconventional in vitro methods (organogenesis, somatic embryogenesis and micrografting) and slow-growth storage conditions for medium-term preservation of Pistacia are presented together with the morphological and molecular studies carried out for the characterization of its species in this review. Moreover, special attention is additionally focused on cryopreservation (dehydration- and vitrification-based one-step freezing techniques) for the long-term preservation of Pistacia species. Possible basic principles concerning the establishment of a cryobank for the successful conservation of Pistacia germplasm are also discussed.     

<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.     

Genetic diversity and structure of the endangered lady's slipper orchid Cypripedium japonicum Thunb. (Orchidaceae) in Japan

Cypripedium japonicum Thunb. (Orchidaceae), once a common perennial herb, is now designated as endangered throughout most of its distribution due to habitat destruction and fragmentation, and the impacts of horticultural collection. We investigated the genetic characteristics of this species for conservation purposes, using microsatellite markers to examine the genetic diversity and structure of 15 native and 5 ex situ populations in Japan. The results imply that although allelic variation is low in Japanese C. japonicum, sexual reproduction by seed, as well as clonal propagation, may occur in some populations. Both native and ex situ populations were found to be genetically differentiated, indicating that some populations may have experienced recent population declines, genetic fragmentation, or bottlenecks. The degree of genetic drift from the putative ancestral population, inferred through STRUCTURE analysis, was more pronounced in northern populations than in southern populations. Some of the ex situ conserved populations exhibited a low degree of differentiation from ancestral native populations. Our results imply that conservation of C. japonicum in Japan is best supported by maintaining individual populations and their unique genetic characteristics.     

Cryoconservation of South African plant genetic diversity

South Africa has a rich flora which exhibits among the highest species density in the world, distributed across nine biomes that support an impressive diversity of animal life. However, a variety of human actions, invasion by alien species, natural disturbances and climate change collectively impact negatively on the great diversity of both plant and animal species. In situ conservation has long been practised, primarily in nature reserves, complemented by ex situ conservation in national botanic gardens, but in vitro plant conservation is not common. In the context of animal biodiversity conservation, the Wildlife Biological Resource Centre of the National Zoological Gardens utilises cryobanking as one of its major focuses and is now poised to expand as the repository for the cryoconservation of plant germplasm, particularly for indigenous recalcitrant-seeded and poor-seeding species. However, there are particular problems associated with successful germplasm cryostorage of such tropical and subtropical plants. As we see the science and application of cryobiology and cryoconservation as cross-cutting and transdisciplinary, we have entrained formal networking among scientists offering a range of specialisations aimed at a deeper understanding of common problems and practical outcomes to facilitate both plant and animal biobanking. The endeavours are aimed at elucidating the basis of both successes and failures in our efforts to attain optimal outcomes. With focus on best practices, standard operating procedures, validation and risk management for cryopreserved and cold-stored plant and animal material, our ultimate aim is to facilitate restoration by the safe reintroduction of indigenous species.     

Ex situ cultivation affects genetic structure and diversity in arable plants

Worldwide, botanical gardens cultivate around 80,000 taxa, corresponding to approximately one‐quarter of all vascular plants. Most cultivated taxa are, however, held in a small number of collections, and mostly only in small populations. Lack of genetic exchange and stochastic processes in small populations make them susceptible to detrimental genetic effects, which should be most severe in annual species, as sowing cycles are often short. In order to assess whether ex situ cultivation affects genetic diversity of annuals, five annual arable species with similar breeding systems were assessed with 42 in situ populations being compared to 20 ex situ populations using a random amplified polymorphic DNA (RAPD) analysis approach. Population sizes tended to be lower under ex situ cultivation and levels of genetic diversity also tended to be lower in four of the five species, with differences being significant in only two. Ex situ populations showed incomplete representation of alleles found in the wild. The duration of cultivation did not indicate any effect on genetic diversity. This implies that cultivation strategies resulted in different genetic structures in the garden populations. Although not unequivocally pronounced, differences nonetheless imply that conservation strategies in the involved gardens may need improvement. One option is cold storage of seeds, a practice that is not currently followed in the studied ex situ collections. This may reflect that the respective gardens focus on displaying living plant populations.     

Conservation of medicinal plants of Western Ghats,India and its sustainable utilization through <Emphasis Type="Italic">in vitro</Emphasis> technology

Climate change, alien species, and use of land for intensive farming and development are causing severe threat to the plant genetic diversity worldwide. Hence, conservation of biodiversity is considered fundamental and also provides the livelihoods to millions of people worldwide. Medicinal plants play a key role in the treatment of a number of diseases, and they are only the source of medicine for majority of people in the developing world. The tropical regions of the world supply the bulk of current global demand for “natural medicine,” albeit with increasing threat to populations in the world and its genetic diversity. India is a major center of origin and diversity of crop and medicinal plants. India poses out 20,000 species of higher plants, one third of it being endemic and 500 species are categorized to have medicinal value. The Western Ghats is one of the major repositories of medicinal plants. It harbors around 4,000 species of higher plants of which 450 species are threatened. Currently, the number of species added to the red list category in this region is increasing, and the valuable genetic resources are being lost at a rapid rate. Demand for medicinal plants is increasing, and this leads to unscrupulous collection from the wild and adulteration of supplies. Providing high-quality planting material for sustainable use and thereby saving the genetic diversity of plants in the wild is important. During the last 25 years of intensive research, Tropical Botanic Garden and Research Institute has developed in vitro protocol for rapid regeneration and establishment of about 40 medicinally important rare and threatened plants of Western Ghats. In situ conservation alone would not be effective in safeguarding these important species. Thus, utilizing the biotechnoligical approach to complement ex situ conservation program is becoming vital. Propagating biotechnology tools in plant conservation program is a prerequisite to succeed in sustainable use and to complement the existing ex situ measures. In addition to propagation, storage of these valuable genetic resources is equally important. In vitro slow growth of 35 species and cryopreservation using embryo/meristem/seed in 20 different species of rare medicinal plants of this region is accomplished. Plants developed in vitro of ten medicinal plants, which have restricted distribution, were reintroduced in the natural habitat as well.     

ISSR Analysis of the Genetic Diversity of the Endangered Species Sinopodophyllum hexandrum （Royle） Ying from Western Sichuan Province, China

Slnopodophyllum hexandrum （Royle） Ying Is an Important medicinal and endangered species. Inter-simple sequence repeats （ISSR） analysis was conducted on seven natural populations from western Slchuan Province to Investigate the genetic diversity of S. hexandrum. Leaf samples of 140 Individuals were collected. Of the 139 discernible fragments generated by 12 selected primers （among 100 primers）, 54 appeared to be polymorphlc. The percentage of polymorphlc bands （PPB） was 38.85% at the species level, and PPB within a population ranged from 7.91% to 23.74%. Low levels of genetic variation （He = 0.092, Ho = 0.142） and high levels of genetic differentiation among the populations （Gst= 62.25%） was detected on the basis of results from POPGENE and analysis of molecular variance （AMOVA）, respectively. Furthermore, the limited gene flow （Nm = 0.361） may result from biological characteristics, such as self-pollination and short distance seed dispersal. Based on the genetic and ecological Information available for S. hexandrum, we propose some appropriate strategies for the conservation of the endangered medicinal species in this region, namely rescuing and conserving the core populations for in situ conservation and sampling and preserving more populations with fewer Individuals from each population for ex situ conservation.     

Genetic diversity and similarity revealed via molecular analysis among and within an in situ population and ex situ accessions of chiltepín (Capsicum annuum var. glabriusculum)

Molecular analysis of genetic diversity amongand within phenotypically similar wild Capsicum annuum var. glabriusculum(chile) populations revealed geneticdifferences among accessions spread over abroad geographic range. These chiles areregionally known as chiltepíns and are a 50metric ton per year wild harvest for the spiceindustry, as well as a genetic resource forcrop improvement. Understanding geneticvariability in this species providesinformation related to conservation efforts. The objective of this research was to surveygenetic diversity among and within an insitu population and ex situ accessionsof chiltepíns. Random AmplifiedPolymorphic DNA (RAPD) molecular markers wereused to study the genetic structure of an in situ population found at the nothernmostrange of this species and ex situaccessions collected from Mexico and Guatemala. Novel genetic variation was found in both thein situ northern disjunct population, aswell as some ex situ accessions, thussupporting conservation of this species viaboth in situ and ex situ strategies The evidence presented here supports effortsto conserve outlier populations via insitu management practices.