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The science and economics of ex situ plant conservation
Review
Special Issue: Plant science research in botanic gardens
The science and economics of ex situ plant conservation De-Zhu Li1 and Hugh W. Pritchard2 1
Plant Germplasm and Genomics Center, Germplasm Bank of Wild Species; and Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan 650204, China 2 Seed Conservation Department, Royal Botanic Gardens, Kew, Wakehurst Place, Ardingly, West Sussex RH17 6TN, UK
Ex situ seed storage underpins global agriculture and food supplies and enables the conservation of thousands of wild species of plants within national and international facilities. As an insurance policy against extinction, ex situ seed conservation is estimated to cost as little as 1% of in situ conservation. The assumptions, costs, risks and scientific challenges associated with ex situ plant conservation depend on the species, the methods employed and the desired storage time. Recent, relatively widespread evidence of less than expected longevity at conventional seed bank temperatures, innovations in the cryopreservation of recalcitrant-seeded species and economic comparators provide compelling evidence that ultra-cold storage should be adopted for the long-term conservation of plants. Policy instruments, such as the Global Strategy for Plant Conservation (2011–2020), should respond to the evidence base and promote the implementation of cryopreservation for both tropical and temperate plants. Plant conservation science and economics Green plants are the primary producers of ecosystems on Earth. However, plant diversity is currently being lost at an unprecedented rate, resulting in an associated decrease in ecosystem services. To address these concerns about plant diversity conservation, the 10-year Global Strategy for Plant Conservation (GSPC) was approved in 2002, establishing priorities for conservation, especially for threatened species. Conservation success is dependent on baseline knowledge and the application of this understanding, whilst considering cost-benefits. Here we review recent developments in plant conservation science and economics in the context of the current GSPC with a view to anticipating essential commitments during its next phase between 2011–2020. Our geographic focus is China, the fastest-growing major nation for the past 30 years, as the impact of economic growth on the environment and its rich biodiversity continues to be immense. The status of global plant diversity Plant diversity is currently being lost at a 100- to 1000-fold higher rate than during the recent geological past [1], resulting in an associated decrease in ecosystem services, Corresponding authors: Li, D.-Z. ([email protected]); Pritchard, H.W. ([email protected]).
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such as the provision of food, fuel, biochemicals and fibre [2]. In an attempt to counter these effects the GSPC was approved in Decision VI/9 of the Conference of the Parties (COP) to the Convention on Biological Diversity on 19 April 2002 in The Hague (http://www.cbd.int/gspc/). Priorities have been set for threatened species and regions (Targets 4, 5, 7, 8, 9) and for the development of protocols and models to enhance the conservation options (Target 3) for both crops and wild species; the remaining ten targets on plant diversity cover understanding, conservation, sustainable use, promotion of education and capacity-building for conservation action [3]. Areas featuring exceptional concentrations of endemic species were identified as biodiversity hotspots and most of these are experiencing exceptional loss of habitat [4]. An estimated 44% of all species of vascular plants is restricted to 25 hotspots, comprising only 1.4% of the Earth’s land surface. Mittermeier et al. [5] now recognize 34 biodiversity hotspots in the world, including the mountains of southwest China. China is megadiverse, with 31000 species of vascular plants, including many ‘living fossils’ that survived the climate changes of the Miocene (23 to 5 MYBP) and the Pleistocene (2.6 MYBP to 12 000 years ago) glaciations [6,7]. Approximately two thirds of the vascular plant species of China are in the southwest, a region that is the most endemic-rich temperate flora of the world [8,9], the centre of distribution of many genera, (e.g. Rhododendron and Primula [10]), and the location of an evergreen broadleaved forest ecosystem dominated by subtropical species of Fagaceae, Lauraceae, Theaceae and Magnoliaceae, whose seed biologies are little known. China has been growing at an average annual GDP growth rate 10%, which continues to exert considerable pressure on natural, including plant genetic, resources. Consequently, conservation actions, both in situ and ex situ, will be of great significance at the global, regional and national level. The completion of Flora Reipublicae Popularis Sinicae [6] and Flora Yunnanica [11], the listing of 1900 species as protected by the State Forestry Administration of China (some of which are illustrated in Figure 1) and the application of the IUCN’s criteria for the conservation status of 4408 species by the Chinese Plant Specialist Group [12], have all contributed significantly to GSPC targets. It is hoped that applications of DNA barcoding techniques in biodiversity hotspots will provide a new tool to document and assess plant diversity in the genomics era [13]. As an
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Figure 1. Seeds of representative species of rare and threatened flora of southwest China. Upper left, seed of Quercus (Cyclobalanopsis) sichourensis (Fagaceae) with only a few individuals in the wild in south Yunnan; Upper right, seed of Schima argentea (Theaceae), a dominant species in south Yunnan; Lower left, seed of Circaeaster agrestis (Kingdoniaceae), a rare and endangered species in northwest Yunnan; Lower right, seed of Psammosilene tunicoides (Caryophyllaceae), a rare, endangered medicinal plant in Yunnan. Photos: Jie Cai (Kunming Institute of Botany, China) and Wolfgang Stuppy (Royal Botanic Gardens, Kew, UK).
official response to the GSPC, China’s Strategy for Plant Conservation declared the nation’s commitment to reversing the national loss of plant diversity [12]. However, biodiversity conservation and economic development seem to be an unresolved conflict in China. Unless the plant heritage of China and other biodiversity-rich regions of the world are preserved, future opportunities to use plants as part of the adaptation strategy to climate change will be significantly diminished. Such biodiversity contains the genetic elements necessary to underpin forthcoming developments in food production, agriculture, forestry and wider ecosystem functioning to cope with abiotic and biotic stresses [14]. Although it is possible to conserve most plant species both in situ and ex situ, there are economic drivers working against in situ conservation and some technical challenges for ex situ conservation. Nonetheless, these conservation approaches should be viewed as complementary rather than alternatives that provide different types of protection for the species. Conservation plans Conservation plans for plants relate to their maintenance either in situ (e.g. protected areas) or ex situ (e.g. botanic gardens, seed banks and tissue culture collections).
In situ conservation In situ conservation, (e.g. natural reserves and conservation corridors), is considered to be an important way of conservation. However, natural and man-made disasters and development (e.g. hydropower, mineral extraction, tourism, land conversion for crops including rubber trees [15], illicit felling and fires) are putting species in natural reserves and in unprotected areas under considerable pressure. Of greatest conservation concern is the fate of long-lived species, as the renewal–replacement cycle can be decades to centuries. Man-made fragmentation is likely to have caused loss of genetic connectivity among populations of Taxus wallichiana var. mairei, an endangered conifer in China [16]. Based on modelling the effect of climate change on the distribution of oak (Quercus) and pine (Pinus) species in Mexico [17], their current distribution is predicted to decrease by 7 - 48% and 0.2 - 64% by 2050, respectively. Predicting the consequences of contracting distribution on species’ survival is the subject of ongoing research. Ex situ conservation Ex situ conservation acts as a back-up for certain segments of diversity that might otherwise be lost in nature and in 615
Review human-dominated ecosystems, generally through the maintenance of clonal crops in field gene banks and in vitro banks, certain trees in conservation stands, and many seed-bearing species in botanic gardens and / or in seed banks (conventional and cryogenic). The call to explore ex situ strategies for key groups of species is long-standing [18], but in practice one approach, seed banking, is used for the maintenance of most collections ex situ [19,20], often to internationally agreed standards [21]. Seeds are relatively easy to collect, can represent a range of genetic diversity in the species if harvested from a population of individuals and can be stored in a relatively small space. Seed banking can also be, and has been, scaled up in terms of facilities (from chest freezer to walk-in cold stores) and effort (i.e. now targeting the conservation of tens of thousands of species). In response to a significant proportion of the world’s flowering plants and crop wild relatives being in jeopardy, conservation practitioners have started to act in concert, targeting threatened, endemic and socio-economically important plants. Conservation biologists have long been concerned for the rich plant and animal biodiversity of southwest China, which are under threat from the rapid economic development in the region. In 1999, Professor Zheng-Yi Wu of the Chinese Academy of Sciences’ Kunming Institute of Botany brought the matter directly to the attention of then Chinese Prime Minister Rongji Zhu. In a partnership with the Millennium Seed Bank (MSB) Project at the Royal Botanic Gardens Kew (http://www.kew.org/msbp/index.htm), the Germplasm Bank of Wild Species (GBOWS; http://www. kib.ac.cn/KIBEnglish/English/index.html) was established in Kunming in January 2007. By September 2009, 31 199 accessions of seeds, mostly collected in southwest China, had been collected for the facility, among which 4 866 species have been identified, including the species shown in Figure 1; the conservation target is 19 000 species in 15 years [22]. In keeping with best practice in genetic resources preservation, the aim is to store duplicate collections on two sites; thus, seeds of some species from southwest China are conserved in the MSB, and 200 UK collections are already conserved in GBOWS. Since 1997, the MSB Project target has been to conserve collaboratively both the UK flora and 10% of the world’s flowering plants (or 24 200 species) by 2010, which will be achieved by the end of 2009, thereby setting a benchmark for future conservation efforts. Ever since humans planted their first crop, they have saved seeds for use in future. In this regard, the largest seed banks have been devoted to crops and other economically important species. The National Center for Genetic Resources Preservation, Fort Collins, Colorado, USA (http://www.ars.usda.gov/main/site_main.htm?modecode= 54-02-05-00), established in 1958, holds a collection of half a million seed accessions, plus the largest collection of apple germplasm in the world. The National Centre for Crop Germplasm Preservation at the Chinese Academy of Agricultural Science in Beijing (http://icgr.caas.net.cn/ cgrisngb.html) is a nationwide network of seed banks that preserves around 400 000 accessions of crop seeds. Partly because of the increasing alarm about climate change and its impact on world food production, the Svalbard 616
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Global Seed Vault (http://www.croptrust.org/main/arctic. php?itemid=211) was built in Norway in February 2008 as a coordinated effort to consolidate and systematize conservation of the world’s crops, particularly those listed on the International Treaty on Plant Genetic Resources for Food and Agriculture (ITPGRFA), which entered into force in 2004 [23]. Before the MSB Project was initiated in 1997, there was little global effort to conserve wild species as seeds. The GBOWS facility is a parallel effort and, more significantly, is built in proximity to some biodiversity hotspots. Although it is accepted that ex situ seed banking and in situ conservation are complementary, a better understanding of this interrelation is needed, as is an appreciation of the role of ex situ conservation in global environmental considerations [24]. Valuing biodiversity and conservation costs Both plants and seeds are a valuable commodity. Global seed sales are estimated to be worth > US$30 billion annually, mainly in support of the agricultural sector [25], and >200 million tonnes of oilseeds (soybean (Glycine), rapeseed (Brassica), groundnut (Arachis) and cottonseed (Gossypium)) are produced each year by the top three producing countries (USA, Brazil and China) [26]. At the species level, such economic assessment is extraordinarily difficult beyond the main crops, particularly for species not yet fully characterised for traits of societal value. Thus, it is difficult to judge what constitutes value for money with respect to the cost of conservation of species in general. However, a current assessment is being made of ‘The Economics of Ecosystems and Biodiversity’ (TEEB) [27]. For example, the estimated cost of preserving one hectare of tropical forest ranges from US$1 to US$27 per annum for Sumatra and Malaysia, respectively [28]. Conservation of the Cape Floristic Region of South Africa would incur both opportunity (e.g. foregone economic development) and management (e.g. fencing and breeding programmes) costs. With a one-time payment of US$522 million, plus annual maintenance expenses of US$24.4 million [29], the cost of conserving this region over 100 years would be US$2900 million. Although this region represents <0.5% of the area of Africa, it contains nearly 20% of the continent’s flora, including 8 920 flowering plant species [30]. Thus to conserve these flowering plants in situ will cost US$ 330 000 (£200 000) per plant species over a 100-year period. Fauna and micro-organisms will also benefit directly from in situ conservation. Nonetheless, an interesting comparison can be made with the costs of ex situ seed conservation within the MSB Project over the past ten years. The average cost for fieldwork, transport, seed processing, banking and viability assessment £2 100 per species (Simon Linington, personal communication). At as little as 1% of the cost of conserving the species in situ, ex situ seed conservation represents excellent value for money as an insurance policy against the irreversible loss of the species from the natural environment. However, not all species have seeds that will store easily for 100 years at conventional seed bank conditions (pre-drying seeds to low moisture content with circa 15% relative humidity air and storage at circa 20 8C) as recognised by the GSPC [3].
Review Risks in the banking sector GSPC Target 8 is ‘60% of threatened plant species in accessible ex situ collections, preferably in the country of origin, and 10% of them included in recovery and restoration programmes’ [3]. There are a number of factors that might reduce successful preservation in ex situ collections below an optimum. These include a failure to develop knowledge and apply technologies appropriately to ensure the preservation of species and an aversion to invest in innovative approaches. Orthodox, bankable seeds, such as the main crops used in mass food and feed production, show, within limits, systematic improvements in longevity on dehydration and cooling [31–33]. By contrast, recalcitrant seeds, such as oaks (Quercus) and cacao (Theobroma), are sensitive to drying [14,34]. The mechanisms by which recalcitrant seeds lose viability are various and complex, including membrane damage and metabolic dysfunction [34]. Interestingly, death in the dry state for orthodox seeds and desiccation-induced death in recalcitrant seeds (and viability loss in other plant cells in response to stress) follow the path of programmed cell death after an initiation step that is modulated by reactive oxygen species-induced oxidation of the major cellular antioxidant and redox buffer, glutathione [35]. Such understanding of the similar means of viability loss under disparate stresses increases the likelihood of developing measures of protective intervention to enable the preservation of biodiverse seeds. Two other recent scientific developments warrant mention here: i) improvements in the rapid diagnosis and prediction of recalcitrant seed responses, and ii) innovations in the cryopreservation of recalcitrant seed tissues. Diagnosis and prediction of desiccation sensitivity in seeds Although there are exceptions, most recalcitrant seeds identified to date are from shrubby species or trees and their frequency is highest (47%) in species of tropical moist forests [36]. As such a habitat probably contains >50% of the world’s plant diversity, it might be that >25% of the world’s species produce recalcitrant seeds. The question is: which ones? Target 3 of the GSPC called for the ‘development of models with protocols for plant conservation and sustainable use, based on research and practical experience’. The presence of interspecific variation in seed storage responses within genera (e.g. coffee (Coffea) and Citrus [37]) shows that there is no simple taxonomic relationship among species exhibiting recalcitrant responses, suggesting that it is a derived trait, with tolerance having been lost several times [34,38]. A general association has been observed between larger seed mass and desiccation sensitivity (e.g. Refs [34,39]), and correlations also sought with seed shape and moisture content at maturity (e.g. in Meliaceae [39]) and a combination of larger seed mass and high precipitation at the time of seed shedding [40]. Relatively thin seed ‘coats’, (i.e. a small seed-coat ratio), which facilitates rapid germination [41], combined with larger seed mass predicted, with high reliability, seed desiccation sensitivity in >100 tree species from Panama, Europe and
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Figure 2. Probabalistic model of seed desiccation sensitivity in relation to seed mass and seed coat ratio (SCR) based on logistic analysis using 104 Panamanian woody species and validated on 38 species from Europe and Africa. With permission of the authors [42]. e3:2699:974aþ2:156b PðDesiccation sensitivityÞ ¼ 1 þ e3:2699:974aþ2:156b where a is SCR and b is log10(seed mass) in grams (g). The equation yields a response curve for every combination of seed mass (0.01–24 g) and SCR (0–1). Seeds with thin coats (SCR <0.2) and a mass of >1 g are highly likely to be desiccation sensitive.
Africa [42] (Figure 2). Future studies should validate this type of modeling for biodiverse species from other floras. Parental environment affects the level of desiccation tolerance and other traits in seeds at seed shedding, but few studies have explored their dependency on measured environmental conditions. In desiccation-sensitive seeds of Aesculus hippocastanum (horse chestnut) across Europe, higher seed mass, (relative) desiccation tolerance and germinability, and lower axis moisture content and solute potential, correlated with heat sum (thermal time) accumulated during development [43]. This provided a quantitative explanation for intraspecific variability in recalcitrant seed traits for this, and possibly, other species. Similar, enhanced seed quality with developmental heat sum occurred in Acer pseudoplatanus (sycamore) fruit growing in its native, southern European rather than more northerly, introduced range [44]. This resulted in reclassification of this species as not recalcitrant and the seeds being cryopreserved after partial drying [45]. The problem remains, however, of how to conserve ex situ a broad range of recalcitrant species; this is probably the greatest conservation science challenge of the 21st century. Meeting the challenge will depend greatly on the further development and much wider application of cryopreservation technology. Cryopreservation comes of age Since the discovery of glycerol as a cryoprotectant 60 years ago, cryoprotectants have become accepted as antifreezes needed to protect life in the frozen state [46,47]. Rather than temperature per se, it is the biophysical changes 617
Review brought about by ice formation, and the associated potentially excessive freeze-dehydration, during cooling that are the main causes of damage, for example to cell membranes and organelles [47]. However, when controlled such freezedehydration concentrates the intra- and inter-cellular water to the point that it forms a glassy (vitreous) matrix, conferring relatively high stability and storability. Innovations in plant cryobiology over the past 25 years include the widescale use of complex mixtures of cryoprotectants that readily form glasses on cooling. Plant vitrification solutions have now been used successfully to cryopreserve shoot tips and other plant tissues (e.g. somatic embryos and nodal explants) from >110 species across >80 genera [48]. Vitrification can be combined with another innovation, encapsulation of tissue in calcium alginate beads, enabling the application of very drastic treatment that would be lethal to naked specimens and facilitating the manipulation of samples [49]. Shoot tips have also been successfully isolated from germinating recalcitrant seeds and cryopreserved via encapsulationvitrification [50]. Excision of tissues is perceived necessarily as an assault with a concomitant oxidative burst, for example superoxide production on isolation of embryo axes, which is then exacerbated during subsequent dehydration [51]. As reactive oxygen species are also involved in cellular signalling and development [49], current studies aim to ‘manage’ the pleiotropic effects of these molecules [51]. Generally, the preferred tissue for the cryopreservation of difficult-to-store seeds is the embryonic axis, which can be rapidly dried to 0.2 g H2O g DW1 before rapid cooling in liquid nitrogen (e.g. in 14 species of Amaryllidaceae [52]). The main advantage is that preservation of the whole axis with functional shoot and root tips simplifies the post-thaw recovery and production of a whole plant. Cryopreservation is the only larger-scale, long-term option for the ex situ conservation of species that are clonal or have recalcitrant seeds. But are misconceptions about the economics of cryopreservation restricting its application? For potato (Solanum tubersosum and related species), the cost of cryopreservation of the shoot tip (plus in vitro culture and soil transfer) is s13–20 per annum per accession, which is approximately one quarter the cost of annual field maintenance (circa s50–60, based on 2002 costs) [53]. Somewhat higher costs of maintenance of an ex situ collection in a botanic garden can be assumed, e.g. >s250 per annum per species (Nigel Taylor, personal communication). Cryopreservation of elm (Ulmus spp.) buds is economically competitive to field clonal archives; based on a comprehensive economic comparison of elm clone conservation as a field gene bank and as 200 dormant buds in cryopreservation, from which direct regrowth is possible, a twofold cost saving was found in favour of the cryobank [54]. For cassava (Cassava spp) clones at the International Centre of Tropical Agriculture (CIAT; http:// www.ciat.cgiar.org/), there are similar cost estimates of US$5 per accession per year for both the field gene bank and as in vitro material, with maintenance in cryopreservation [55]. Maintenance of the fruit tree germplasm under cryopreservation cost the USDA up to US$75 per accession for initial transfer and US$1 per year for liquid nitrogen supply [55]. 618
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Similarly, the cost of maintaining (i.e. carrying over to next year) orthodox seeds of wheat (Triticum spp.) and maize (Zea spp.) in the seed bank of the Centro Internacional de Mejoramiento de Maiz y Trigo (CIMMYT; http:// www.cimmyt.org/) in Mexico in 2001 was US$0.19 and US$0.93, respectively, or up to US$30 to store an accession for the 40-year life of the seed bank [56]. Storing soybean seed at 6% moisture and 15 8C is estimated to cost about US$1 per year over a 100-year period [57]. However, the initial costs for the incorporation of a newly acquired accession of wheat seeds of initial low quality that requires regeneration in the first year increases to US$8 [56,58], and viability monitoring costs could amount to 20–25% of the total preservation costs [57]. Similar maintenance costs of 20% of the collection and initial preservation cost can be expected for species held in the Millennium Seed Bank over a 100-year period, that is, <£5 per species per annum, excluding reinstallation costs for cold rooms (Simon Linington, personal communication). One of the projected benefits of liquid nitrogen storage is the long-term deferment of regeneration costs, as specimens will have ‘indefinite’ lifespans. But no biological sample is immortal. After 28 years of cryopreservation, 59% of the 91 strawberry meristems thawed were viable compared to 56% viability after eight weeks cryopreservation [59]. Although only 14% of 78 pea meristems thawed were viable after 28 years compared with 61% after 26 weeks of cryopreservation, the highest viability from a single ampoule was 77% [59]. Moreover, cryopreservation of orthodox seeds after the onset of viability loss in dry storage was not able to stop a fall in quality [60]. Nonetheless, cryogenic storage did prolong the shelf life of lettuce (Lactuca) seeds with projected half-lives in the vapour and liquid phases of liquid nitrogen of 500 and 3 400 years, respectively [60], up to 20 times greater than that predicted for that species in a conventional seed bank at 20 8C [32,33]. When orthodoxy is not enough Recovery of plants from orthodox, desiccation tolerant seeds is projected to be possible after centuries to millennia in seed banks, based on the seed viability equations [31– 33], apparently removing the need to consider cryopreservation as an ex situ option for such material. Predicting seed longevity for the world flora (242 000 species) is challenging, although indications are that species adapted to hotter, dry environments have evolved longer lifespans in the dry state. For example, Orobanche crenata and O. aegyptiaca (broomrapes), mainly from southern Europe, North Africa and the Middle East, are relatively long-lived compared with lettuce and barley (Hordeum) seed [see 61]. Also, maximum annual temperatures between 25 8C and 35 8C for the collection location of the seeds has been correlated with survival of dry heat (103 8C) for 17 hours, in many species of Aizoaceae and Cactaceae, but not of Crassulaceae [62]. Moreover, a few seeds are known to have survived for >200 years (e.g. for a few species from the drier regions of NE China [63] and the Cape Floristic Region [64]), and for 2000 years, for a date palm from Israel [65]. Similarly, across nearly 200 species, it was found that species from drier (total rainfall) and warmer temperature
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considered here have seeds that tolerate cryopreservation, a technique that also lends itself to the preservation of mycorrhizal fungi [75], which are important for the successful establishment of plants in the natural environment.
Figure 3. Spread of half-life (P50) estimates for seeds of 276 species mostly stored for 24–26 years at 18 8C after earlier storage at 5 8C. Only 61 (22%) and 147 (53%) of species have seeds predicted to realise half-lives of >100 and >50 years respectively under long-term storage conditions. Data adapted from [67]. As the number of species with seed lifespans of >100 years under conventional seed bank conditions is potentially limited, cryopreservation in liquid nitrogen of a sublot of all orthodox seed samples is recommended, as an additional insurance policy against species extinction.
(mean annual) locations tended to have greater seed P50 (time taken in storage for viability to fall to 50%) under accelerated ageing conditions than species from cool, wet conditions [66]. Moreover, species P50 values were correlated with the proportion of collections (not necessarily the same species) in that family that lost a significant amount of viability after 20 years under conditions for long term seed storage, that is, seeds pre-equilibrated with 15% relative humidity air and then stored at 20 8C [66], as accepted by the international community [21]. Such relative underperformance at 20 8C was apparent in 26% of collections [66]. Similarly, it has been estimated that half-lives for the seeds of 276 species held for an average of 38 years under cool (5 8C) and cold (25 years at 18 8C) temperature was >100 years only for 61 (22%) of the species [67] (Figure 3). Although 25 species (from 19 genera) of Cruciferae had high germination (often >90%) after nearly 40 years storage at 5 to 10 8C [68], it is hard not to conclude that, as an extra insurance policy for conservation, cryopreservation should be considered appropriate for all orthodox seeds. Evidence for unusually poor seed longevity at 20 8C was made 30 years ago, when it was revealed that the storage performance of partially aged barley seeds in the gene bank was equivalent to storage at approximately 6 8C [69]. Evidence later emerged of rapid viability loss at 20 8C and 0 8C in dry seeds of some tropical species, mainly oilseeds, such as coffee (e.g. Ref. [70]). Similar sensitivity to dry storage in the gene bank for Cattleya aurantiaca (orange cattleya) [71] and Cuphea spp. (waxweed) [72,73] was proposed to be associated with conformational changes in the seed lipids, as determined by differential scanning calorimetry. As composition determines the temperature of lipid phase changes and varies between species, cold sensitivity among species might be expected to vary. Evidence for this has been presented for some orchid seeds with sensitivity to storage at 30 8C and 50 8C [74]. Importantly, many of the species
Conclusions and future directions We have highlighted the need to increase our efforts at developing ex situ conservation approaches for plants, particularly those from biodiversity hotspots and with recalcitrant seeds. Undoubtedly, cryopreservation will become increasingly important in the delivery of this goal and in enabling the long-term preservation of orthodox seeds. Concerns about the costs of ex situ conservation compared with in situ have been allayed, and ex situ conservation revealed to be excellent value for money. Consequently, we believe that a relaunched Target 8 of the GSPC (2011–2020) should set clear aims for the use of cryopreserevation to protect threatened and vulnerable species. Other scientific and management objectives need addressing under Target 8. For example, how to create self-sustaining populations of threatened species [76,77], what capacity the soil seed bank has to restore genetic potential [78], and improvements in the regulatory environment for the provision of material for restoration [79]. Without developments in these areas there will be many more cases similar to that of Metasequoia glyptostroboides (dawn redwood); an extensive programme of increasing the number of individuals and distribution range for the species has not enabled the restoration of the genetic structure of this living fossil from China [80]. Scientists, government departments and non-governmental organisations [12,76,81,82] increasingly appreciate seed banking as an effective and economic conservation tool because of its complementarity to in situ approaches [83]. But it is not necessarily a feature of the science curriculum. By combining studies of the world’s biodiversity hotspots, particularly the remaining tropical rainforests in the Afrotropic, Australasian and Indo-Malayan (Sundaland) regions [Rhett A. Butler, A place out of time: tropical rainforests – Their wonders and the perils they face, http://rainforests.mongabay.com/about.htm] with an enhanced effort to train young scientists in, inter alia, taxonomy and seed biology, we could enable sustained conservation activities for decades to come. Acknowledgements We thank our colleagues, Xiang-Yun Yang, Jie Cai, Lulu Huang and Wolfgang Stuppy for various support. This study was supported by the National Basic Research Programme of China (973 Programme, grant number: 2007CB411600), the Ministry of Science and Technology of China (grant number: 2005DKA21006) and the Millennium Seed Bank Project of the Royal Botanic Gardens, Kew, UK, which receives grant-inaid from the Defra, UK.
References 1 Corvalan, C., Hales, S. and McMichael, A. (2005) Millennium Ecosystem Assessment (2005). Ecosystems and Human Well-being: Biodiversity Synthesis, World Resources Institute, Washington, DC, USA 2 European Communities (2008) The Economics of Ecosystems and Biodiversity. An interim Report, European Communities ISBN-13 978-92-79-08960-2 619
Review 3 CBD (2002), Global Strategy for Plant Conservation, The Secretariat of the Convention on Biological Diversity, Montreal, Canada 4 Myers, N. et al. (2000) Biodiversity hotspots for conservation priorities. Nature 403, 853–858 ISBN 0 9539141 5 1 5 Mittermeier, R.A. et al. (2005) Hotspots Revisited: Earth’s Biologically Richest and Most Endangered Terrestrial Ecoregions, The University of Chicago Press, Chicago 6 Wu, C-Y. et al. (2004) Flora Reipiblicaae Popularis Sinicae (Vol. 1), Science Press, Beijing, China 7 Li, D-Z. (2008) Floristics and plant biogeography in China. J. Integr. Plant Biol. 50, 771–777 8 Wang, W-T. et al. (1993) Vascular Plants of the Hengduan Mountains 1, Science Press, Beijing, China 9 Wang, W-T. et al. (1993) Vascular Plants of the Hengduan Mountains 2, Science Press, Beijing, China 10 Wu, Z-Y. (1988) Hengduan Mountain flora and her significance. J. Jap. Bot. 63, 297–311 11 Chu, W-M. et al. (2006) Flora Yunnanica, tom. 20, Science Press, Beijing, China 12 CSPC Editorial-Committee (2008) China’s Strategy for Plant Conservation, Guangdong Press Group, Guangdong Science and Technology Press, Guangzhou, China 13 Lahaye, R. et al. (2008) DNA barcoding the floras of biodiversity hotspots. Proc. Nat. Acad. Sci. U.S.A. 105, 2923–2928 14 Black, M. and Pritchard, H.W., eds (2002) Desiccation and Survival in Plants. Drying Without Dying, CABI Publishing, Wallingford, UK 15 Ziegler, A.D. et al. (2009) The rubber juggernaut. Science 324, 1024– 1025 16 Zhang, X-M. et al. (2009) Molecular evidence for fragmentation among populations of Taxus wallichiana var. mairei, a highly endangered conifer in China. Can. J. For. Res. 39, 755–764 17 Go´mez-Mendoza, L. and Arriaga, L. (2007) Modelling the effect of climate change on the distribution of oak and pine species of Mexico. Conserv. Biol. 21, 1545–1555 18 Roche, L. (1975) The Methodology of Conservation of Forest Genetic Resources. Report on a Pilot Study, FAO 19 FAO (1997) The State of the World’s Plant Genetic Resources for Food and Agriculture, FAO 20 Linington, S.H. and Pritchard, H.W. (2001) Genebanks. In Encyclopaedia of Biodiversity, Volume 3, (S.A. Levin, ed.), pp. 165– 181, Academic Press, San Francisco 21 FAO and IPGRI (1994) Genebank Standards, FAO and IPGRI, Rome, p. 13, ISBN 92-9043-236-5 22 Cyranoski, D. (2003) Biodiversity schemes take root in China. Nature 425, 890 23 FAO (2009) A Global Treaty for Food Security and Sustainable Agriculture. International Treaty on Plant Genetic Resources for Food and Agriculture, FAO 24 Cohen, J.I. et al. (1991) Ex situ conservation of plant genetic resources: global development and environmental concerns. Science 253, 866–872 25 International Seed Federation (2008) Estimated value of the domestic seed market in selected countries, International Seed Federation, (Switzerland) 26 The Economist (2006) Pocket World in Figures, 2007 Edition, The Economist Newspapers 27 European Communities (2008). The Economics of Ecosystems and Biodiversity. An Interim Report, 68 pp, ISBN-13 978-92-79-08960-2, European Communities 28 Wilson, K.A. et al. (2005) Measuring and incorporating vulnerability into conservation planning. Environ. Manag. 35, 527–543 29 Frazee, S.R. et al. (2003) Estimating the costs of conserving a biodiversity hotspot: a case-study of the Cape Floristic Region, South Africa. Biol. Conserv. 112, 275–290 30 Goldblatt, P. and Manning, J.C. (2002) Plant diversity of the Cape Region of South Africa. Ann. Miss. Bot. Gard. 89, 281–302 31 Ellis, R.H. and Roberts, E.H. (1980) Improved equations for the prediction of seed longevity. Ann. Bot. 45, 13–30 32 Roberts, E.H. and Ellis, R.H. (1989) Water and seed survival. Ann. Bot. 63, 39–52 33 Dickie, J.B. et al. (1990) Temperature and seed storage longevity. Ann. Bot. 65, 197–204 34 Berjak, P. and Pammenter, N.W. (2007) From Avicennia to Zizania: seed recalcitrance in perspective. Ann. Bot. 101, 213–228
620
Trends in Plant Science Vol.14 No.11 35 Kranner, I. et al. (2006) Glutathione half-cell reduction potential: a universal stress marker and modulator of programmed cell death? Free Rad. Biol. Med. 40, 2155–2165 36 Tweddle, J.C. et al. (2003) Ecological aspects of seed desiccation sensitivity. J. Ecol. 91, 294–304 37 Hong, T.D. and Ellis, R.H. (1995) Interspecific variation in seed storage behaviour within two genera - Coffea and Citrus. Seed Sci. Technol. 23, 165–181 38 Dickie, J.B. and Pritchard, H.W. (2002) Systematic and evolutionary aspects of desiccation tolerance in seeds. In Desiccation and Survival in Plants: Drying Without Dying (Black, M. and Pritchard, H.W., eds), pp. 239–259, Wallingford, UK, CABI Publishing 39 Hong, T.D. and Ellis, R.H. (1998) Contrasting seed storage behaviour among different species of Meliaceae. Seed Sci. Technol. 26, 77–95 40 Pritchard, H.W. et al. (2004) Ecological correlates of seed desiccation tolerance in tropical dryland trees. Am. J. Bot. 91, 863–870 41 Daws, M.I. et al. (2006) Traits of recalcitrant seeds in a semi-deciduous tropical forest in Panama: some ecological implications. Funct. Ecol. 19, 874–885 42 Daws, M.I. et al. (2006) Prediction of desiccation sensitivity in seeds of woody species: a probabilistic model based on two seed traits and 104 species. Ann. Bot. 97, 667–674 43 Daws, M.I. et al. (2004) Developmental heat sum influences recalcitrant seed traits in Aesculus hippocastanum across Europe. New Phytol. 162, 157–166 44 Daws, M.I. et al. (2006) Variable desiccation tolerance in Acer pseudoplatanus seeds in relation to developmental conditions: a case of phenotypic recalcitrance? Funct. Plant Biol. 33, 59–66 45 Daws, M.I. and Pritchard, H.W. (2008) The development and limits of freezing tolerance in Acer pseudoplatanus fruits across Europe is dependent on provenance. CryoLetters 30, 189–198 & 527 46 Fuller, B.J. et al. (2004) Life in the Frozen State, CRC Press 47 Fuller, B.J. (2004) Cryoprotectants: the essential antifreezes to protect life in the frozen state. CryoLetters 25, 375–388 48 Sakai, A. and Engelmann, F. (2007) Vitrification, encapsulationvitrification and droplet-vitrification: a review. CryoLetters 28, 151–172 49 Benson, E.E. (2008) Cryopreservation of phytodiversity: a critical appraisal of theory and practice. Critical Rev. Plant Sci. 27, 141–219 50 Nadarajan, J. et al. (2008) Application of differential scanning calorimetry in developing cryopreservation strategies for Parkia speciosa: a tropical tree producing recalcitrant seeds. CryoLetters 29, 95–110 51 Roach, T. et al. (2008) An oxidative burst of superoxide in embryonic axes of recalcitrant sweet chestnut seeds as induced by excision and desiccation. Physiol. Plant. 133, 131–139 52 Sershen et al. (2007) Cryopreservation of embryonic axes of selected amaryllid species. CryoLetters 28, 387–399 53 Keller, E.R.J. et al. (2008) Cryopreservation for plant genebanks: a matter between high expectations and cautious reservation. CryoLetters 29, 53–62 54 Harvengt, L. et al. (2004) Establishment of a cryopreserved genebank of European elms. Can. J. For. Res. 34, 43–55 55 Reed, B.M. et al. (2004) Technical Guidelines for the Management of Field and In Vitro Germplasm Collections, IPGRI Handbooks for Genebanks, No. 7. IPGRI (now Biodiversity International) 56 Pardey, P.G. et al. (2001) Costing the ex situ conservation of genetic resources: maize and wheat at CIMMYT. Crop Sci. 41, 1286–1299 57 Walters, C. (2003) Optimising seed banking procedures. In Seed Conservation: Turning Science into Practice (Smith, R.D. et al., eds), pp. 723–743, Kew, UK, Royal Botanic Gardens 58 Koo, B. and Smale, B. (2003) Economic costs of genebank operations. In A Guide to Effective Management of Germplasm Collections (IPGRI Handbook for Genebanks No 6) (Engels, J.M.M. and Visser, L., eds), IPGRI (now Bioversity International) 59 Caswell, K.L. and Kartha, K.K. (2009) Recovery of plants from pea and strawberry meristems cryopreserved for 28 years. CryoLetters 30, 41–46 60 Walters, C. et al. (2004) Longevity of cryogenically stored seeds. Cryobiology 48, 229–244 61 Pritchard, H.W. and Dickie, J.B. (2003) Predicting seed longevity: the use and abuse of seed viability equations. In Seed Conservation: Turning Science into Practice (Smith, R.D. et al., eds), pp. 653–721, Kew, UK, Royal Botanic Gardens
Review 62 Daws, M.I. et al. (2007) Extreme thermo-tolerance in seeds of desert succulents is related to maximum annual temperature. S. Afr. J. Bot. 73, 262–265 63 Shen-Miller, J. et al. (1995) Exceptional seed longevity and robust growth: ancient sacred lotus from China. Am. J. Bot. 82, 1367–1380 64 Daws, M.I. et al. (2007) Two-hundred-year seed survival of Leucospermum, and two other woody species from the Cape Floristic Region, South Africa. Seed Sci. Res. 17, 73–79 65 Sallon, S. et al. (2008) Germination, genetics, and growth of an ancient date seed. Science 320, 1464 66 Probert, R.J. et al. (2009) Ecological correlates of ex situ seed longevity: a comparative study on 195 species. Ann. Bot. 104, 57–69 67 Walters, C. et al. (2005) Longevity of seeds stored in a genebank: species characteristics. Seed Sci. Res. 15, 1–20 68 Perez-Garcia, F. et al. (2007) High viability recorded in ultra-dry seeds of 37 species of Brassicaceae after almost 40 years of storage. Seed Sci. Technol. 35, 143–155 69 Roberts, E.H. and Ellis, R.H. (1977) Prediction of seed longevity at subzero temperatures and genetic resources conservation. Nature 268, 431–433 70 Ellis, R.H. et al. (1990) An intermediate category of seed storage behaviour? I. Coffee. J. Exp. Bot. 41, 1167–1174 71 Pritchard, H.W. and Seaton, P.T. (1993) Orchid seed storage: historical perspective, current status and future prospects. Selbyana 14, 89–104 72 Crane, J. et al. (2003) Triacylglycerols determine the unusual storage physiology of Cuphea seed. Planta 217, 699–708 73 Crane, J. et al. (2006) Triacylglycerol phase and ‘intermediate’ seed storage physiology: a study of Cuphea carthagenesis. Planta 223, 1081–1089
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74 Pritchard, H.W. et al. (1999) Interspecific variation in orchid seed longevity in relation to ultra-drying and cryopreservation. Lindleyana 14, 92–101 75 Wood, C.B. et al. (2000) Simultaneous preservation of orchid seed and its fungal symbiont using encapsulation-dehydration is dependent on moisture content and storage temperature. CryoLetters 21, 125–136 76 Cochrane, J.A. et al. (2007) The significance of ex situ seed conservation to reintroduction of threatened plants. Aust. J. Bot. 55, 356–361 77 Guerrant, E.O.J. and Kaye, T.N. (2007) Reintroduction of rare and endangered plants: common factors, questions and approaches. Austr. J. Bot. 55, 362–370 78 Uesugi, R. et al. (2007) Restoration of genetic diversity from soil seed banks in a threatened aquatic plant, Nymphoides peltata. Conserv. Genet. 8, 111–121 79 Smith, S.L. et al. (2007) Genetic diversity in restoration materials and the impacts of seed collection in Colorado’s restoration plant production industry. Rest. Ecol. 15, 369–374 80 Li, Y-Y. et al. (2005) Genetic differences between wild and artificial populations of Metasequoia glyptostroboides: implications for species recovery. Conser. Biol. 19, 224–231 81 Bo¨rner, A. (2006) Preservation of plant genetic resources in the biotechnology era. Biotechnol. J. 1, 1393–1404 82 Prada, D. (2009) Molecular population genetics and agronomic alleles in seed banks: searching for a needle in a haystack? J. Exp. Bot. 60, 2541–2552 83 Maxted, N. et al. (1997) Complementary conservation strategies. In Plant Genetic Conservation: The In Situ Approach (Maxted, N., FordLloyd, B.V. and Hawkes, J.G., eds), pp. 15–39, Chapman and Hall
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Special Issue: Plant science research in botanic gardens
The science and economics of ex situ plant conservation De-Zhu Li1 and Hugh W. Pritchard2 1
Plant Germplasm and Genomics Center, Germplasm Bank of Wild Species; and Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan 650204, China 2 Seed Conservation Department, Royal Botanic Gardens, Kew, Wakehurst Place, Ardingly, West Sussex RH17 6TN, UK
Ex situ seed storage underpins global agriculture and food supplies and enables the conservation of thousands of wild species of plants within national and international facilities. As an insurance policy against extinction, ex situ seed conservation is estimated to cost as little as 1% of in situ conservation. The assumptions, costs, risks and scientific challenges associated with ex situ plant conservation depend on the species, the methods employed and the desired storage time. Recent, relatively widespread evidence of less than expected longevity at conventional seed bank temperatures, innovations in the cryopreservation of recalcitrant-seeded species and economic comparators provide compelling evidence that ultra-cold storage should be adopted for the long-term conservation of plants. Policy instruments, such as the Global Strategy for Plant Conservation (2011–2020), should respond to the evidence base and promote the implementation of cryopreservation for both tropical and temperate plants. Plant conservation science and economics Green plants are the primary producers of ecosystems on Earth. However, plant diversity is currently being lost at an unprecedented rate, resulting in an associated decrease in ecosystem services. To address these concerns about plant diversity conservation, the 10-year Global Strategy for Plant Conservation (GSPC) was approved in 2002, establishing priorities for conservation, especially for threatened species. Conservation success is dependent on baseline knowledge and the application of this understanding, whilst considering cost-benefits. Here we review recent developments in plant conservation science and economics in the context of the current GSPC with a view to anticipating essential commitments during its next phase between 2011–2020. Our geographic focus is China, the fastest-growing major nation for the past 30 years, as the impact of economic growth on the environment and its rich biodiversity continues to be immense. The status of global plant diversity Plant diversity is currently being lost at a 100- to 1000-fold higher rate than during the recent geological past [1], resulting in an associated decrease in ecosystem services, Corresponding authors: Li, D.-Z. ([email protected]); Pritchard, H.W. ([email protected]).
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such as the provision of food, fuel, biochemicals and fibre [2]. In an attempt to counter these effects the GSPC was approved in Decision VI/9 of the Conference of the Parties (COP) to the Convention on Biological Diversity on 19 April 2002 in The Hague (http://www.cbd.int/gspc/). Priorities have been set for threatened species and regions (Targets 4, 5, 7, 8, 9) and for the development of protocols and models to enhance the conservation options (Target 3) for both crops and wild species; the remaining ten targets on plant diversity cover understanding, conservation, sustainable use, promotion of education and capacity-building for conservation action [3]. Areas featuring exceptional concentrations of endemic species were identified as biodiversity hotspots and most of these are experiencing exceptional loss of habitat [4]. An estimated 44% of all species of vascular plants is restricted to 25 hotspots, comprising only 1.4% of the Earth’s land surface. Mittermeier et al. [5] now recognize 34 biodiversity hotspots in the world, including the mountains of southwest China. China is megadiverse, with 31000 species of vascular plants, including many ‘living fossils’ that survived the climate changes of the Miocene (23 to 5 MYBP) and the Pleistocene (2.6 MYBP to 12 000 years ago) glaciations [6,7]. Approximately two thirds of the vascular plant species of China are in the southwest, a region that is the most endemic-rich temperate flora of the world [8,9], the centre of distribution of many genera, (e.g. Rhododendron and Primula [10]), and the location of an evergreen broadleaved forest ecosystem dominated by subtropical species of Fagaceae, Lauraceae, Theaceae and Magnoliaceae, whose seed biologies are little known. China has been growing at an average annual GDP growth rate 10%, which continues to exert considerable pressure on natural, including plant genetic, resources. Consequently, conservation actions, both in situ and ex situ, will be of great significance at the global, regional and national level. The completion of Flora Reipublicae Popularis Sinicae [6] and Flora Yunnanica [11], the listing of 1900 species as protected by the State Forestry Administration of China (some of which are illustrated in Figure 1) and the application of the IUCN’s criteria for the conservation status of 4408 species by the Chinese Plant Specialist Group [12], have all contributed significantly to GSPC targets. It is hoped that applications of DNA barcoding techniques in biodiversity hotspots will provide a new tool to document and assess plant diversity in the genomics era [13]. As an
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Figure 1. Seeds of representative species of rare and threatened flora of southwest China. Upper left, seed of Quercus (Cyclobalanopsis) sichourensis (Fagaceae) with only a few individuals in the wild in south Yunnan; Upper right, seed of Schima argentea (Theaceae), a dominant species in south Yunnan; Lower left, seed of Circaeaster agrestis (Kingdoniaceae), a rare and endangered species in northwest Yunnan; Lower right, seed of Psammosilene tunicoides (Caryophyllaceae), a rare, endangered medicinal plant in Yunnan. Photos: Jie Cai (Kunming Institute of Botany, China) and Wolfgang Stuppy (Royal Botanic Gardens, Kew, UK).
official response to the GSPC, China’s Strategy for Plant Conservation declared the nation’s commitment to reversing the national loss of plant diversity [12]. However, biodiversity conservation and economic development seem to be an unresolved conflict in China. Unless the plant heritage of China and other biodiversity-rich regions of the world are preserved, future opportunities to use plants as part of the adaptation strategy to climate change will be significantly diminished. Such biodiversity contains the genetic elements necessary to underpin forthcoming developments in food production, agriculture, forestry and wider ecosystem functioning to cope with abiotic and biotic stresses [14]. Although it is possible to conserve most plant species both in situ and ex situ, there are economic drivers working against in situ conservation and some technical challenges for ex situ conservation. Nonetheless, these conservation approaches should be viewed as complementary rather than alternatives that provide different types of protection for the species. Conservation plans Conservation plans for plants relate to their maintenance either in situ (e.g. protected areas) or ex situ (e.g. botanic gardens, seed banks and tissue culture collections).
In situ conservation In situ conservation, (e.g. natural reserves and conservation corridors), is considered to be an important way of conservation. However, natural and man-made disasters and development (e.g. hydropower, mineral extraction, tourism, land conversion for crops including rubber trees [15], illicit felling and fires) are putting species in natural reserves and in unprotected areas under considerable pressure. Of greatest conservation concern is the fate of long-lived species, as the renewal–replacement cycle can be decades to centuries. Man-made fragmentation is likely to have caused loss of genetic connectivity among populations of Taxus wallichiana var. mairei, an endangered conifer in China [16]. Based on modelling the effect of climate change on the distribution of oak (Quercus) and pine (Pinus) species in Mexico [17], their current distribution is predicted to decrease by 7 - 48% and 0.2 - 64% by 2050, respectively. Predicting the consequences of contracting distribution on species’ survival is the subject of ongoing research. Ex situ conservation Ex situ conservation acts as a back-up for certain segments of diversity that might otherwise be lost in nature and in 615
Review human-dominated ecosystems, generally through the maintenance of clonal crops in field gene banks and in vitro banks, certain trees in conservation stands, and many seed-bearing species in botanic gardens and / or in seed banks (conventional and cryogenic). The call to explore ex situ strategies for key groups of species is long-standing [18], but in practice one approach, seed banking, is used for the maintenance of most collections ex situ [19,20], often to internationally agreed standards [21]. Seeds are relatively easy to collect, can represent a range of genetic diversity in the species if harvested from a population of individuals and can be stored in a relatively small space. Seed banking can also be, and has been, scaled up in terms of facilities (from chest freezer to walk-in cold stores) and effort (i.e. now targeting the conservation of tens of thousands of species). In response to a significant proportion of the world’s flowering plants and crop wild relatives being in jeopardy, conservation practitioners have started to act in concert, targeting threatened, endemic and socio-economically important plants. Conservation biologists have long been concerned for the rich plant and animal biodiversity of southwest China, which are under threat from the rapid economic development in the region. In 1999, Professor Zheng-Yi Wu of the Chinese Academy of Sciences’ Kunming Institute of Botany brought the matter directly to the attention of then Chinese Prime Minister Rongji Zhu. In a partnership with the Millennium Seed Bank (MSB) Project at the Royal Botanic Gardens Kew (http://www.kew.org/msbp/index.htm), the Germplasm Bank of Wild Species (GBOWS; http://www. kib.ac.cn/KIBEnglish/English/index.html) was established in Kunming in January 2007. By September 2009, 31 199 accessions of seeds, mostly collected in southwest China, had been collected for the facility, among which 4 866 species have been identified, including the species shown in Figure 1; the conservation target is 19 000 species in 15 years [22]. In keeping with best practice in genetic resources preservation, the aim is to store duplicate collections on two sites; thus, seeds of some species from southwest China are conserved in the MSB, and 200 UK collections are already conserved in GBOWS. Since 1997, the MSB Project target has been to conserve collaboratively both the UK flora and 10% of the world’s flowering plants (or 24 200 species) by 2010, which will be achieved by the end of 2009, thereby setting a benchmark for future conservation efforts. Ever since humans planted their first crop, they have saved seeds for use in future. In this regard, the largest seed banks have been devoted to crops and other economically important species. The National Center for Genetic Resources Preservation, Fort Collins, Colorado, USA (http://www.ars.usda.gov/main/site_main.htm?modecode= 54-02-05-00), established in 1958, holds a collection of half a million seed accessions, plus the largest collection of apple germplasm in the world. The National Centre for Crop Germplasm Preservation at the Chinese Academy of Agricultural Science in Beijing (http://icgr.caas.net.cn/ cgrisngb.html) is a nationwide network of seed banks that preserves around 400 000 accessions of crop seeds. Partly because of the increasing alarm about climate change and its impact on world food production, the Svalbard 616
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Global Seed Vault (http://www.croptrust.org/main/arctic. php?itemid=211) was built in Norway in February 2008 as a coordinated effort to consolidate and systematize conservation of the world’s crops, particularly those listed on the International Treaty on Plant Genetic Resources for Food and Agriculture (ITPGRFA), which entered into force in 2004 [23]. Before the MSB Project was initiated in 1997, there was little global effort to conserve wild species as seeds. The GBOWS facility is a parallel effort and, more significantly, is built in proximity to some biodiversity hotspots. Although it is accepted that ex situ seed banking and in situ conservation are complementary, a better understanding of this interrelation is needed, as is an appreciation of the role of ex situ conservation in global environmental considerations [24]. Valuing biodiversity and conservation costs Both plants and seeds are a valuable commodity. Global seed sales are estimated to be worth > US$30 billion annually, mainly in support of the agricultural sector [25], and >200 million tonnes of oilseeds (soybean (Glycine), rapeseed (Brassica), groundnut (Arachis) and cottonseed (Gossypium)) are produced each year by the top three producing countries (USA, Brazil and China) [26]. At the species level, such economic assessment is extraordinarily difficult beyond the main crops, particularly for species not yet fully characterised for traits of societal value. Thus, it is difficult to judge what constitutes value for money with respect to the cost of conservation of species in general. However, a current assessment is being made of ‘The Economics of Ecosystems and Biodiversity’ (TEEB) [27]. For example, the estimated cost of preserving one hectare of tropical forest ranges from US$1 to US$27 per annum for Sumatra and Malaysia, respectively [28]. Conservation of the Cape Floristic Region of South Africa would incur both opportunity (e.g. foregone economic development) and management (e.g. fencing and breeding programmes) costs. With a one-time payment of US$522 million, plus annual maintenance expenses of US$24.4 million [29], the cost of conserving this region over 100 years would be US$2900 million. Although this region represents <0.5% of the area of Africa, it contains nearly 20% of the continent’s flora, including 8 920 flowering plant species [30]. Thus to conserve these flowering plants in situ will cost US$ 330 000 (£200 000) per plant species over a 100-year period. Fauna and micro-organisms will also benefit directly from in situ conservation. Nonetheless, an interesting comparison can be made with the costs of ex situ seed conservation within the MSB Project over the past ten years. The average cost for fieldwork, transport, seed processing, banking and viability assessment £2 100 per species (Simon Linington, personal communication). At as little as 1% of the cost of conserving the species in situ, ex situ seed conservation represents excellent value for money as an insurance policy against the irreversible loss of the species from the natural environment. However, not all species have seeds that will store easily for 100 years at conventional seed bank conditions (pre-drying seeds to low moisture content with circa 15% relative humidity air and storage at circa 20 8C) as recognised by the GSPC [3].
Review Risks in the banking sector GSPC Target 8 is ‘60% of threatened plant species in accessible ex situ collections, preferably in the country of origin, and 10% of them included in recovery and restoration programmes’ [3]. There are a number of factors that might reduce successful preservation in ex situ collections below an optimum. These include a failure to develop knowledge and apply technologies appropriately to ensure the preservation of species and an aversion to invest in innovative approaches. Orthodox, bankable seeds, such as the main crops used in mass food and feed production, show, within limits, systematic improvements in longevity on dehydration and cooling [31–33]. By contrast, recalcitrant seeds, such as oaks (Quercus) and cacao (Theobroma), are sensitive to drying [14,34]. The mechanisms by which recalcitrant seeds lose viability are various and complex, including membrane damage and metabolic dysfunction [34]. Interestingly, death in the dry state for orthodox seeds and desiccation-induced death in recalcitrant seeds (and viability loss in other plant cells in response to stress) follow the path of programmed cell death after an initiation step that is modulated by reactive oxygen species-induced oxidation of the major cellular antioxidant and redox buffer, glutathione [35]. Such understanding of the similar means of viability loss under disparate stresses increases the likelihood of developing measures of protective intervention to enable the preservation of biodiverse seeds. Two other recent scientific developments warrant mention here: i) improvements in the rapid diagnosis and prediction of recalcitrant seed responses, and ii) innovations in the cryopreservation of recalcitrant seed tissues. Diagnosis and prediction of desiccation sensitivity in seeds Although there are exceptions, most recalcitrant seeds identified to date are from shrubby species or trees and their frequency is highest (47%) in species of tropical moist forests [36]. As such a habitat probably contains >50% of the world’s plant diversity, it might be that >25% of the world’s species produce recalcitrant seeds. The question is: which ones? Target 3 of the GSPC called for the ‘development of models with protocols for plant conservation and sustainable use, based on research and practical experience’. The presence of interspecific variation in seed storage responses within genera (e.g. coffee (Coffea) and Citrus [37]) shows that there is no simple taxonomic relationship among species exhibiting recalcitrant responses, suggesting that it is a derived trait, with tolerance having been lost several times [34,38]. A general association has been observed between larger seed mass and desiccation sensitivity (e.g. Refs [34,39]), and correlations also sought with seed shape and moisture content at maturity (e.g. in Meliaceae [39]) and a combination of larger seed mass and high precipitation at the time of seed shedding [40]. Relatively thin seed ‘coats’, (i.e. a small seed-coat ratio), which facilitates rapid germination [41], combined with larger seed mass predicted, with high reliability, seed desiccation sensitivity in >100 tree species from Panama, Europe and
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Figure 2. Probabalistic model of seed desiccation sensitivity in relation to seed mass and seed coat ratio (SCR) based on logistic analysis using 104 Panamanian woody species and validated on 38 species from Europe and Africa. With permission of the authors [42]. e3:2699:974aþ2:156b PðDesiccation sensitivityÞ ¼ 1 þ e3:2699:974aþ2:156b where a is SCR and b is log10(seed mass) in grams (g). The equation yields a response curve for every combination of seed mass (0.01–24 g) and SCR (0–1). Seeds with thin coats (SCR <0.2) and a mass of >1 g are highly likely to be desiccation sensitive.
Africa [42] (Figure 2). Future studies should validate this type of modeling for biodiverse species from other floras. Parental environment affects the level of desiccation tolerance and other traits in seeds at seed shedding, but few studies have explored their dependency on measured environmental conditions. In desiccation-sensitive seeds of Aesculus hippocastanum (horse chestnut) across Europe, higher seed mass, (relative) desiccation tolerance and germinability, and lower axis moisture content and solute potential, correlated with heat sum (thermal time) accumulated during development [43]. This provided a quantitative explanation for intraspecific variability in recalcitrant seed traits for this, and possibly, other species. Similar, enhanced seed quality with developmental heat sum occurred in Acer pseudoplatanus (sycamore) fruit growing in its native, southern European rather than more northerly, introduced range [44]. This resulted in reclassification of this species as not recalcitrant and the seeds being cryopreserved after partial drying [45]. The problem remains, however, of how to conserve ex situ a broad range of recalcitrant species; this is probably the greatest conservation science challenge of the 21st century. Meeting the challenge will depend greatly on the further development and much wider application of cryopreservation technology. Cryopreservation comes of age Since the discovery of glycerol as a cryoprotectant 60 years ago, cryoprotectants have become accepted as antifreezes needed to protect life in the frozen state [46,47]. Rather than temperature per se, it is the biophysical changes 617
Review brought about by ice formation, and the associated potentially excessive freeze-dehydration, during cooling that are the main causes of damage, for example to cell membranes and organelles [47]. However, when controlled such freezedehydration concentrates the intra- and inter-cellular water to the point that it forms a glassy (vitreous) matrix, conferring relatively high stability and storability. Innovations in plant cryobiology over the past 25 years include the widescale use of complex mixtures of cryoprotectants that readily form glasses on cooling. Plant vitrification solutions have now been used successfully to cryopreserve shoot tips and other plant tissues (e.g. somatic embryos and nodal explants) from >110 species across >80 genera [48]. Vitrification can be combined with another innovation, encapsulation of tissue in calcium alginate beads, enabling the application of very drastic treatment that would be lethal to naked specimens and facilitating the manipulation of samples [49]. Shoot tips have also been successfully isolated from germinating recalcitrant seeds and cryopreserved via encapsulationvitrification [50]. Excision of tissues is perceived necessarily as an assault with a concomitant oxidative burst, for example superoxide production on isolation of embryo axes, which is then exacerbated during subsequent dehydration [51]. As reactive oxygen species are also involved in cellular signalling and development [49], current studies aim to ‘manage’ the pleiotropic effects of these molecules [51]. Generally, the preferred tissue for the cryopreservation of difficult-to-store seeds is the embryonic axis, which can be rapidly dried to 0.2 g H2O g DW1 before rapid cooling in liquid nitrogen (e.g. in 14 species of Amaryllidaceae [52]). The main advantage is that preservation of the whole axis with functional shoot and root tips simplifies the post-thaw recovery and production of a whole plant. Cryopreservation is the only larger-scale, long-term option for the ex situ conservation of species that are clonal or have recalcitrant seeds. But are misconceptions about the economics of cryopreservation restricting its application? For potato (Solanum tubersosum and related species), the cost of cryopreservation of the shoot tip (plus in vitro culture and soil transfer) is s13–20 per annum per accession, which is approximately one quarter the cost of annual field maintenance (circa s50–60, based on 2002 costs) [53]. Somewhat higher costs of maintenance of an ex situ collection in a botanic garden can be assumed, e.g. >s250 per annum per species (Nigel Taylor, personal communication). Cryopreservation of elm (Ulmus spp.) buds is economically competitive to field clonal archives; based on a comprehensive economic comparison of elm clone conservation as a field gene bank and as 200 dormant buds in cryopreservation, from which direct regrowth is possible, a twofold cost saving was found in favour of the cryobank [54]. For cassava (Cassava spp) clones at the International Centre of Tropical Agriculture (CIAT; http:// www.ciat.cgiar.org/), there are similar cost estimates of US$5 per accession per year for both the field gene bank and as in vitro material, with maintenance in cryopreservation [55]. Maintenance of the fruit tree germplasm under cryopreservation cost the USDA up to US$75 per accession for initial transfer and US$1 per year for liquid nitrogen supply [55]. 618
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Similarly, the cost of maintaining (i.e. carrying over to next year) orthodox seeds of wheat (Triticum spp.) and maize (Zea spp.) in the seed bank of the Centro Internacional de Mejoramiento de Maiz y Trigo (CIMMYT; http:// www.cimmyt.org/) in Mexico in 2001 was US$0.19 and US$0.93, respectively, or up to US$30 to store an accession for the 40-year life of the seed bank [56]. Storing soybean seed at 6% moisture and 15 8C is estimated to cost about US$1 per year over a 100-year period [57]. However, the initial costs for the incorporation of a newly acquired accession of wheat seeds of initial low quality that requires regeneration in the first year increases to US$8 [56,58], and viability monitoring costs could amount to 20–25% of the total preservation costs [57]. Similar maintenance costs of 20% of the collection and initial preservation cost can be expected for species held in the Millennium Seed Bank over a 100-year period, that is, <£5 per species per annum, excluding reinstallation costs for cold rooms (Simon Linington, personal communication). One of the projected benefits of liquid nitrogen storage is the long-term deferment of regeneration costs, as specimens will have ‘indefinite’ lifespans. But no biological sample is immortal. After 28 years of cryopreservation, 59% of the 91 strawberry meristems thawed were viable compared to 56% viability after eight weeks cryopreservation [59]. Although only 14% of 78 pea meristems thawed were viable after 28 years compared with 61% after 26 weeks of cryopreservation, the highest viability from a single ampoule was 77% [59]. Moreover, cryopreservation of orthodox seeds after the onset of viability loss in dry storage was not able to stop a fall in quality [60]. Nonetheless, cryogenic storage did prolong the shelf life of lettuce (Lactuca) seeds with projected half-lives in the vapour and liquid phases of liquid nitrogen of 500 and 3 400 years, respectively [60], up to 20 times greater than that predicted for that species in a conventional seed bank at 20 8C [32,33]. When orthodoxy is not enough Recovery of plants from orthodox, desiccation tolerant seeds is projected to be possible after centuries to millennia in seed banks, based on the seed viability equations [31– 33], apparently removing the need to consider cryopreservation as an ex situ option for such material. Predicting seed longevity for the world flora (242 000 species) is challenging, although indications are that species adapted to hotter, dry environments have evolved longer lifespans in the dry state. For example, Orobanche crenata and O. aegyptiaca (broomrapes), mainly from southern Europe, North Africa and the Middle East, are relatively long-lived compared with lettuce and barley (Hordeum) seed [see 61]. Also, maximum annual temperatures between 25 8C and 35 8C for the collection location of the seeds has been correlated with survival of dry heat (103 8C) for 17 hours, in many species of Aizoaceae and Cactaceae, but not of Crassulaceae [62]. Moreover, a few seeds are known to have survived for >200 years (e.g. for a few species from the drier regions of NE China [63] and the Cape Floristic Region [64]), and for 2000 years, for a date palm from Israel [65]. Similarly, across nearly 200 species, it was found that species from drier (total rainfall) and warmer temperature
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considered here have seeds that tolerate cryopreservation, a technique that also lends itself to the preservation of mycorrhizal fungi [75], which are important for the successful establishment of plants in the natural environment.
Figure 3. Spread of half-life (P50) estimates for seeds of 276 species mostly stored for 24–26 years at 18 8C after earlier storage at 5 8C. Only 61 (22%) and 147 (53%) of species have seeds predicted to realise half-lives of >100 and >50 years respectively under long-term storage conditions. Data adapted from [67]. As the number of species with seed lifespans of >100 years under conventional seed bank conditions is potentially limited, cryopreservation in liquid nitrogen of a sublot of all orthodox seed samples is recommended, as an additional insurance policy against species extinction.
(mean annual) locations tended to have greater seed P50 (time taken in storage for viability to fall to 50%) under accelerated ageing conditions than species from cool, wet conditions [66]. Moreover, species P50 values were correlated with the proportion of collections (not necessarily the same species) in that family that lost a significant amount of viability after 20 years under conditions for long term seed storage, that is, seeds pre-equilibrated with 15% relative humidity air and then stored at 20 8C [66], as accepted by the international community [21]. Such relative underperformance at 20 8C was apparent in 26% of collections [66]. Similarly, it has been estimated that half-lives for the seeds of 276 species held for an average of 38 years under cool (5 8C) and cold (25 years at 18 8C) temperature was >100 years only for 61 (22%) of the species [67] (Figure 3). Although 25 species (from 19 genera) of Cruciferae had high germination (often >90%) after nearly 40 years storage at 5 to 10 8C [68], it is hard not to conclude that, as an extra insurance policy for conservation, cryopreservation should be considered appropriate for all orthodox seeds. Evidence for unusually poor seed longevity at 20 8C was made 30 years ago, when it was revealed that the storage performance of partially aged barley seeds in the gene bank was equivalent to storage at approximately 6 8C [69]. Evidence later emerged of rapid viability loss at 20 8C and 0 8C in dry seeds of some tropical species, mainly oilseeds, such as coffee (e.g. Ref. [70]). Similar sensitivity to dry storage in the gene bank for Cattleya aurantiaca (orange cattleya) [71] and Cuphea spp. (waxweed) [72,73] was proposed to be associated with conformational changes in the seed lipids, as determined by differential scanning calorimetry. As composition determines the temperature of lipid phase changes and varies between species, cold sensitivity among species might be expected to vary. Evidence for this has been presented for some orchid seeds with sensitivity to storage at 30 8C and 50 8C [74]. Importantly, many of the species
Conclusions and future directions We have highlighted the need to increase our efforts at developing ex situ conservation approaches for plants, particularly those from biodiversity hotspots and with recalcitrant seeds. Undoubtedly, cryopreservation will become increasingly important in the delivery of this goal and in enabling the long-term preservation of orthodox seeds. Concerns about the costs of ex situ conservation compared with in situ have been allayed, and ex situ conservation revealed to be excellent value for money. Consequently, we believe that a relaunched Target 8 of the GSPC (2011–2020) should set clear aims for the use of cryopreserevation to protect threatened and vulnerable species. Other scientific and management objectives need addressing under Target 8. For example, how to create self-sustaining populations of threatened species [76,77], what capacity the soil seed bank has to restore genetic potential [78], and improvements in the regulatory environment for the provision of material for restoration [79]. Without developments in these areas there will be many more cases similar to that of Metasequoia glyptostroboides (dawn redwood); an extensive programme of increasing the number of individuals and distribution range for the species has not enabled the restoration of the genetic structure of this living fossil from China [80]. Scientists, government departments and non-governmental organisations [12,76,81,82] increasingly appreciate seed banking as an effective and economic conservation tool because of its complementarity to in situ approaches [83]. But it is not necessarily a feature of the science curriculum. By combining studies of the world’s biodiversity hotspots, particularly the remaining tropical rainforests in the Afrotropic, Australasian and Indo-Malayan (Sundaland) regions [Rhett A. Butler, A place out of time: tropical rainforests – Their wonders and the perils they face, http://rainforests.mongabay.com/about.htm] with an enhanced effort to train young scientists in, inter alia, taxonomy and seed biology, we could enable sustained conservation activities for decades to come. Acknowledgements We thank our colleagues, Xiang-Yun Yang, Jie Cai, Lulu Huang and Wolfgang Stuppy for various support. This study was supported by the National Basic Research Programme of China (973 Programme, grant number: 2007CB411600), the Ministry of Science and Technology of China (grant number: 2005DKA21006) and the Millennium Seed Bank Project of the Royal Botanic Gardens, Kew, UK, which receives grant-inaid from the Defra, UK.
References 1 Corvalan, C., Hales, S. and McMichael, A. (2005) Millennium Ecosystem Assessment (2005). Ecosystems and Human Well-being: Biodiversity Synthesis, World Resources Institute, Washington, DC, USA 2 European Communities (2008) The Economics of Ecosystems and Biodiversity. An interim Report, European Communities ISBN-13 978-92-79-08960-2 619
Review 3 CBD (2002), Global Strategy for Plant Conservation, The Secretariat of the Convention on Biological Diversity, Montreal, Canada 4 Myers, N. et al. (2000) Biodiversity hotspots for conservation priorities. Nature 403, 853–858 ISBN 0 9539141 5 1 5 Mittermeier, R.A. et al. (2005) Hotspots Revisited: Earth’s Biologically Richest and Most Endangered Terrestrial Ecoregions, The University of Chicago Press, Chicago 6 Wu, C-Y. et al. (2004) Flora Reipiblicaae Popularis Sinicae (Vol. 1), Science Press, Beijing, China 7 Li, D-Z. (2008) Floristics and plant biogeography in China. J. Integr. Plant Biol. 50, 771–777 8 Wang, W-T. et al. (1993) Vascular Plants of the Hengduan Mountains 1, Science Press, Beijing, China 9 Wang, W-T. et al. (1993) Vascular Plants of the Hengduan Mountains 2, Science Press, Beijing, China 10 Wu, Z-Y. (1988) Hengduan Mountain flora and her significance. J. Jap. Bot. 63, 297–311 11 Chu, W-M. et al. (2006) Flora Yunnanica, tom. 20, Science Press, Beijing, China 12 CSPC Editorial-Committee (2008) China’s Strategy for Plant Conservation, Guangdong Press Group, Guangdong Science and Technology Press, Guangzhou, China 13 Lahaye, R. et al. (2008) DNA barcoding the floras of biodiversity hotspots. Proc. Nat. Acad. Sci. U.S.A. 105, 2923–2928 14 Black, M. and Pritchard, H.W., eds (2002) Desiccation and Survival in Plants. Drying Without Dying, CABI Publishing, Wallingford, UK 15 Ziegler, A.D. et al. (2009) The rubber juggernaut. Science 324, 1024– 1025 16 Zhang, X-M. et al. (2009) Molecular evidence for fragmentation among populations of Taxus wallichiana var. mairei, a highly endangered conifer in China. Can. J. For. Res. 39, 755–764 17 Go´mez-Mendoza, L. and Arriaga, L. (2007) Modelling the effect of climate change on the distribution of oak and pine species of Mexico. Conserv. Biol. 21, 1545–1555 18 Roche, L. (1975) The Methodology of Conservation of Forest Genetic Resources. Report on a Pilot Study, FAO 19 FAO (1997) The State of the World’s Plant Genetic Resources for Food and Agriculture, FAO 20 Linington, S.H. and Pritchard, H.W. (2001) Genebanks. In Encyclopaedia of Biodiversity, Volume 3, (S.A. Levin, ed.), pp. 165– 181, Academic Press, San Francisco 21 FAO and IPGRI (1994) Genebank Standards, FAO and IPGRI, Rome, p. 13, ISBN 92-9043-236-5 22 Cyranoski, D. (2003) Biodiversity schemes take root in China. Nature 425, 890 23 FAO (2009) A Global Treaty for Food Security and Sustainable Agriculture. International Treaty on Plant Genetic Resources for Food and Agriculture, FAO 24 Cohen, J.I. et al. (1991) Ex situ conservation of plant genetic resources: global development and environmental concerns. Science 253, 866–872 25 International Seed Federation (2008) Estimated value of the domestic seed market in selected countries, International Seed Federation, (Switzerland) 26 The Economist (2006) Pocket World in Figures, 2007 Edition, The Economist Newspapers 27 European Communities (2008). The Economics of Ecosystems and Biodiversity. An Interim Report, 68 pp, ISBN-13 978-92-79-08960-2, European Communities 28 Wilson, K.A. et al. (2005) Measuring and incorporating vulnerability into conservation planning. Environ. Manag. 35, 527–543 29 Frazee, S.R. et al. (2003) Estimating the costs of conserving a biodiversity hotspot: a case-study of the Cape Floristic Region, South Africa. Biol. Conserv. 112, 275–290 30 Goldblatt, P. and Manning, J.C. (2002) Plant diversity of the Cape Region of South Africa. Ann. Miss. Bot. Gard. 89, 281–302 31 Ellis, R.H. and Roberts, E.H. (1980) Improved equations for the prediction of seed longevity. Ann. Bot. 45, 13–30 32 Roberts, E.H. and Ellis, R.H. (1989) Water and seed survival. Ann. Bot. 63, 39–52 33 Dickie, J.B. et al. (1990) Temperature and seed storage longevity. Ann. Bot. 65, 197–204 34 Berjak, P. and Pammenter, N.W. (2007) From Avicennia to Zizania: seed recalcitrance in perspective. Ann. Bot. 101, 213–228
620
Trends in Plant Science Vol.14 No.11 35 Kranner, I. et al. (2006) Glutathione half-cell reduction potential: a universal stress marker and modulator of programmed cell death? Free Rad. Biol. Med. 40, 2155–2165 36 Tweddle, J.C. et al. (2003) Ecological aspects of seed desiccation sensitivity. J. Ecol. 91, 294–304 37 Hong, T.D. and Ellis, R.H. (1995) Interspecific variation in seed storage behaviour within two genera - Coffea and Citrus. Seed Sci. Technol. 23, 165–181 38 Dickie, J.B. and Pritchard, H.W. (2002) Systematic and evolutionary aspects of desiccation tolerance in seeds. In Desiccation and Survival in Plants: Drying Without Dying (Black, M. and Pritchard, H.W., eds), pp. 239–259, Wallingford, UK, CABI Publishing 39 Hong, T.D. and Ellis, R.H. (1998) Contrasting seed storage behaviour among different species of Meliaceae. Seed Sci. Technol. 26, 77–95 40 Pritchard, H.W. et al. (2004) Ecological correlates of seed desiccation tolerance in tropical dryland trees. Am. J. Bot. 91, 863–870 41 Daws, M.I. et al. (2006) Traits of recalcitrant seeds in a semi-deciduous tropical forest in Panama: some ecological implications. Funct. Ecol. 19, 874–885 42 Daws, M.I. et al. (2006) Prediction of desiccation sensitivity in seeds of woody species: a probabilistic model based on two seed traits and 104 species. Ann. Bot. 97, 667–674 43 Daws, M.I. et al. (2004) Developmental heat sum influences recalcitrant seed traits in Aesculus hippocastanum across Europe. New Phytol. 162, 157–166 44 Daws, M.I. et al. (2006) Variable desiccation tolerance in Acer pseudoplatanus seeds in relation to developmental conditions: a case of phenotypic recalcitrance? Funct. Plant Biol. 33, 59–66 45 Daws, M.I. and Pritchard, H.W. (2008) The development and limits of freezing tolerance in Acer pseudoplatanus fruits across Europe is dependent on provenance. CryoLetters 30, 189–198 & 527 46 Fuller, B.J. et al. (2004) Life in the Frozen State, CRC Press 47 Fuller, B.J. (2004) Cryoprotectants: the essential antifreezes to protect life in the frozen state. CryoLetters 25, 375–388 48 Sakai, A. and Engelmann, F. (2007) Vitrification, encapsulationvitrification and droplet-vitrification: a review. CryoLetters 28, 151–172 49 Benson, E.E. (2008) Cryopreservation of phytodiversity: a critical appraisal of theory and practice. Critical Rev. Plant Sci. 27, 141–219 50 Nadarajan, J. et al. (2008) Application of differential scanning calorimetry in developing cryopreservation strategies for Parkia speciosa: a tropical tree producing recalcitrant seeds. CryoLetters 29, 95–110 51 Roach, T. et al. (2008) An oxidative burst of superoxide in embryonic axes of recalcitrant sweet chestnut seeds as induced by excision and desiccation. Physiol. Plant. 133, 131–139 52 Sershen et al. (2007) Cryopreservation of embryonic axes of selected amaryllid species. CryoLetters 28, 387–399 53 Keller, E.R.J. et al. (2008) Cryopreservation for plant genebanks: a matter between high expectations and cautious reservation. CryoLetters 29, 53–62 54 Harvengt, L. et al. (2004) Establishment of a cryopreserved genebank of European elms. Can. J. For. Res. 34, 43–55 55 Reed, B.M. et al. (2004) Technical Guidelines for the Management of Field and In Vitro Germplasm Collections, IPGRI Handbooks for Genebanks, No. 7. IPGRI (now Biodiversity International) 56 Pardey, P.G. et al. (2001) Costing the ex situ conservation of genetic resources: maize and wheat at CIMMYT. Crop Sci. 41, 1286–1299 57 Walters, C. (2003) Optimising seed banking procedures. In Seed Conservation: Turning Science into Practice (Smith, R.D. et al., eds), pp. 723–743, Kew, UK, Royal Botanic Gardens 58 Koo, B. and Smale, B. (2003) Economic costs of genebank operations. In A Guide to Effective Management of Germplasm Collections (IPGRI Handbook for Genebanks No 6) (Engels, J.M.M. and Visser, L., eds), IPGRI (now Bioversity International) 59 Caswell, K.L. and Kartha, K.K. (2009) Recovery of plants from pea and strawberry meristems cryopreserved for 28 years. CryoLetters 30, 41–46 60 Walters, C. et al. (2004) Longevity of cryogenically stored seeds. Cryobiology 48, 229–244 61 Pritchard, H.W. and Dickie, J.B. (2003) Predicting seed longevity: the use and abuse of seed viability equations. In Seed Conservation: Turning Science into Practice (Smith, R.D. et al., eds), pp. 653–721, Kew, UK, Royal Botanic Gardens
Review 62 Daws, M.I. et al. (2007) Extreme thermo-tolerance in seeds of desert succulents is related to maximum annual temperature. S. Afr. J. Bot. 73, 262–265 63 Shen-Miller, J. et al. (1995) Exceptional seed longevity and robust growth: ancient sacred lotus from China. Am. J. Bot. 82, 1367–1380 64 Daws, M.I. et al. (2007) Two-hundred-year seed survival of Leucospermum, and two other woody species from the Cape Floristic Region, South Africa. Seed Sci. Res. 17, 73–79 65 Sallon, S. et al. (2008) Germination, genetics, and growth of an ancient date seed. Science 320, 1464 66 Probert, R.J. et al. (2009) Ecological correlates of ex situ seed longevity: a comparative study on 195 species. Ann. Bot. 104, 57–69 67 Walters, C. et al. (2005) Longevity of seeds stored in a genebank: species characteristics. Seed Sci. Res. 15, 1–20 68 Perez-Garcia, F. et al. (2007) High viability recorded in ultra-dry seeds of 37 species of Brassicaceae after almost 40 years of storage. Seed Sci. Technol. 35, 143–155 69 Roberts, E.H. and Ellis, R.H. (1977) Prediction of seed longevity at subzero temperatures and genetic resources conservation. Nature 268, 431–433 70 Ellis, R.H. et al. (1990) An intermediate category of seed storage behaviour? I. Coffee. J. Exp. Bot. 41, 1167–1174 71 Pritchard, H.W. and Seaton, P.T. (1993) Orchid seed storage: historical perspective, current status and future prospects. Selbyana 14, 89–104 72 Crane, J. et al. (2003) Triacylglycerols determine the unusual storage physiology of Cuphea seed. Planta 217, 699–708 73 Crane, J. et al. (2006) Triacylglycerol phase and ‘intermediate’ seed storage physiology: a study of Cuphea carthagenesis. Planta 223, 1081–1089
Trends in Plant Science
Vol.14 No.11
74 Pritchard, H.W. et al. (1999) Interspecific variation in orchid seed longevity in relation to ultra-drying and cryopreservation. Lindleyana 14, 92–101 75 Wood, C.B. et al. (2000) Simultaneous preservation of orchid seed and its fungal symbiont using encapsulation-dehydration is dependent on moisture content and storage temperature. CryoLetters 21, 125–136 76 Cochrane, J.A. et al. (2007) The significance of ex situ seed conservation to reintroduction of threatened plants. Aust. J. Bot. 55, 356–361 77 Guerrant, E.O.J. and Kaye, T.N. (2007) Reintroduction of rare and endangered plants: common factors, questions and approaches. Austr. J. Bot. 55, 362–370 78 Uesugi, R. et al. (2007) Restoration of genetic diversity from soil seed banks in a threatened aquatic plant, Nymphoides peltata. Conserv. Genet. 8, 111–121 79 Smith, S.L. et al. (2007) Genetic diversity in restoration materials and the impacts of seed collection in Colorado’s restoration plant production industry. Rest. Ecol. 15, 369–374 80 Li, Y-Y. et al. (2005) Genetic differences between wild and artificial populations of Metasequoia glyptostroboides: implications for species recovery. Conser. Biol. 19, 224–231 81 Bo¨rner, A. (2006) Preservation of plant genetic resources in the biotechnology era. Biotechnol. J. 1, 1393–1404 82 Prada, D. (2009) Molecular population genetics and agronomic alleles in seed banks: searching for a needle in a haystack? J. Exp. Bot. 60, 2541–2552 83 Maxted, N. et al. (1997) Complementary conservation strategies. In Plant Genetic Conservation: The In Situ Approach (Maxted, N., FordLloyd, B.V. and Hawkes, J.G., eds), pp. 15–39, Chapman and Hall
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