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The Role of Botanic Gardens in Ex Situ Conservation
Abstract
Only a green world, rich in plants, can sustain us and the millions of other species with which we share this planet. But, in an era of global change, nature is on the retreat. Like the communities they form, many plant species are becoming rarer, threatened even to the point of extinction. The worldwide community of almost three thousand botanic gardens are holders of the most diverse living collections of plants and have the unique potential to conserve plant diversity. Conservation biology is a fast moving and often controversial field, and, as the contributions within these pages from experts in the field demonstrate, plant conservation is multifaceted, mirroring the complexity of the biodiversity it aims to protect, and striving not just to protect threatened plants but to preserve ecosystem services and secure the integrity of the biosphere.
... Almost two of every five known plant species are threatened with extinction due to large-scale anthropogenic pressures such as land use conversion and climate change (Lughadha et al., 2020). Given these challenges, ex situ living collections are often needed to support in situ conservation efforts in multiple ways (IUCN/SSC, 2014), including producing stock for supplementing wild populations, providing founders for conservation translocation (IUCN/SSC, 2013) or serving as safeguards against extinction in the wild (Smith and Pence, 2017). Securing a species ex situ may entail a living collection of 60 to >200 individuals, if representation of a high proportion of intraspecific variation across the geographic range of the species is required . ...
... Among these kinds of living collections, seed banks may often be most efficient at representing intraspecific variation, because a large number of viable individuals can be stored long-term in small spaces (Smith and Pence, 2017). However, seed banks as well as living collections of whole growing plants, seem to commonly fail at capturing and preserving the variation present in wild populations (Wei and Jiang, 2021). ...
... In contrast to the previous case, spatially separated living collections of plants sourced from a single region are not deemed substitutable, and each region must be represented at each living collection in the metacollection. Doing so increases the resilience of the metacollection to catastrophic events that might affect a particular living collection, including fires and events associated with large-scale disasters, infrastructure failure or socioeconomic stress (Smith and Pence, 2017;Griffith et al., 2019). Because these events are largely unpredictable and rare, they are not accounted for in background survival probabilities, S(t) rc (but see Griffith et al., 2017). ...
... Ex situ plant collections play an increasingly important role in biodiversity conservation amid growing anthropogenic pressures (Oldfield, 2009;Smith and Pence, 2017), including overharvesting, habitat alteration and climate change (Lughadha et al., 2020). Ex situ conservation may be critical to avoid the extinction of rare or threatened plant species when their habitat is heavily degraded or fragmented (Whitlock et al., 2016). ...
... Ex situ conservation may be critical to avoid the extinction of rare or threatened plant species when their habitat is heavily degraded or fragmented (Whitlock et al., 2016). In extreme cases, ex situ conservation may be the only way to prevent species extinction (Smith and Pence, 2017). Often the best practice entails the integration of ex situ and in situ procedures for species recovery, reintroduction and ecological restoration (Newton and Oldfield, 2012). ...
... Collectively, botanic gardens manage at least 105,634 species, including 41% of the species classified as threatened (Mounce et al., 2017). These institutions employ various kinds of ex situ collections, characterized by distinct advantages and limitations (Abeli et al., 2020;Smith and Pence, 2017). Among them, seed banking is the most cost effective and space efficient manner of ex situ conservation for many species (Abeli et al., 2020). ...
Living collections grown in outdoor landscapes within botanic gardens play an increasingly important role in ex situ species conservation, but there is little work predicting the survival of different plant species in these landscapes. Here we present an approach to predict the survival of plants grown from wild propagules in botanic gardens, based on climatic provenance. We developed mixed effects Cox models to relate plant survival to horticulturally informed measures of climatic differences between botanic garden landscapes and the wild distribution of plants. We fit these models to data on the survival of 1184 plantings of 410 species grown in the Missouri Botanical Garden (MBG, in Saint Louis, Missouri, USA) from wild propagules collected around the world. We detected major effects of growth form, minimum temperature of the coldest month, maximum temperature of the warmest month, and to a lesser extent precipitation of the driest month. Survival at MBG was predicted better by the climate of propagule collection localities than by the overall climatic distribution of species, suggesting local adaptation. However, there was substantial interspecific variation in survival among plants from similar climates. The best models predicted high survival for plants grown at MBG from propagules collected within broad latitudinal bands across non-tropical regions. We explore implications of these findings for the role of botanic gardens in ex situ conservation, and discuss the applicability of the approach we developed to enhance the capacity of living collections to represent species of conservation concern.
... Ex situ plant conservation has been widely understood as a crucial policy action, and botanic gardens have a vital role in conducting these efforts. Botanic gardens have been effective in preserving efforts and increasing the diversity of the plant [5]. Botanic gardens are ex situ plant conservation areas with a documented collection of plants arranged based on taxonomic, bioregional, or thematic classification patterns, or a combination of these patterns, for conservation, research, education, tourism activities environmental services [6]. ...
Agathis borneensis , Castanopsis argentea , Diospyros celebica , Merrillia caloxylon , and Saurauia bracteosa are some threatened species, which have been planted in Cibodas Botanic Gardens as a garden collection. As part of the plant conservation program, their vegetative and reproductive phenology knowledge is crucial to understand when to harvest the seeds used for further propagation and restoration. The study aimed to investigate the phenology phases of these species as well as the impact of microclimates. The study used a descriptive-quantitative analysis based on the records of the exploratory-inventory observation of flowering and fruiting phase data and microclimate circumstances in 2018. Except for precipitation, the data revealed that CBG’s microclimate was considerably less diversified. The results also described that A. borneensis has flowering and fruiting almost throughout the year. C. argentea flowers at the end of the year and then develop into fruit at the beginning of the next subsequent year. D. celebica is also a low-intensity flowering plant that bears fruit almost all year. S. bracteosa has a long period of flowering from May to the end of the year. And then it started to develop into fruits from July to the end of the year and continued to January of the following year. Unfortunately, M. caloxylon is not shown a reproductive stage throughout the year. According to the findings, reproductive phases prominently occur early and at the end of the year, along with the rainy season. It also implied the appropriate time for seed harvesting conducted during these periods.
... Botanic gardens' ex situ plant conservation has proven to be successful in preserving and enhancing plant biodiversity. Plant cultivation, seed banking, tissue culture, cryopreservation, species recovery, species conservation status assessment, assisted migration, and ecological restoration are some of the ex situ and in situ conservation strategies used by botanic gardens [1] [2]. In Indonesia, botanic gardens also have some challenges in mitigating plant species extinction. ...
Despite the routinely conducted treatment and maintenance of garden plant collection, the monthly mortality figures of the Cibodas Botanic Gardens (CBG) plant collections remain significant. It is presumed that the microclimate has a crucial influence on plant survivorship in the field. This study aimed to analyze the effect of microclimate conditions on the mortality rate of the CBG plant collection. The study was conducted by correlating the number of mortal plants with CBG monthly microclimatic conditions from 2017 to 2018. The analyzed microclimate parameters were temperature, relative humidity, precipitation, wind velocity, and solar radiation. The multiple regression modeling, t-test, and Pearson correlation test (r) were utilized to measure the level of significance of the correlation (α = 0.05). The findings were shown that the maximum wind velocity was the primary unit correlated to the number of mortality. The correlation was strong positive (r = 68.8%), and significant (p-value α). We suggested anticipating the disadvantages influences of strong wind supported by other extreme microclimate units, such as heavy rain. These events frequently caused high damage to the tree and other plant collections. These are expected to be taken into a consideration by the CBG operator and management in order to forecast and mitigate the risks of future plant collection losses.
... What in vitro methods are needed? Choosing the most effective method requiring the least input of time will allow efficient use of resources and help ensure the conservation of more species (Pence et al., 2020;Smith & Pence, 2017). While the cryogenic genebank is a more recent addition to the conservation toolbox, recognition of the need for increased global capacity is growing, and the Exceptional Plant Conservation Network was established to focus attention on species that cannot be conserved in conventional seed banks (https://cinci nnati zoo.org/epcn). ...
Societal Impact Statement
Eroding plant diversity has serious implications for the well‐being of humanity and our planet. Conserving plants ex situ requires technologies that are rapidly advancing and readily accessible. The alarming loss of plant habitats has spawned global investment in technologies that focus on either conventional freezer storage, which exploit seed adaptations to survive drying, or on cryogenic platforms, that ensure long‐term survival of germplasm that is not amenable to conventional methods. Increasing evidence that germplasm survives for decades provide proof of concept, but also warns of the limited utility of stored germplasm that is not returned to the Earth.
Summary
A future sustainable world requires concerted efforts to conserve plant biodiversity. Using an integrated approach, botanic gardens, arboreta, universities, governmental agencies, and non‐governmental organizations are addressing that challenge. Here, we summarize some of the technological advances, in an ever‐growing toolbox, that increase the scope of taxa that are conserved ex situ as well as the lifespans of diverse plant tissues that can be used as germplasm. Seed banking continues to be a powerful and efficient tool. Seeds that tolerate extreme drying and low temperature will likely survive at least 100 years using conventional conditions of a common freezer. The extreme tolerance of seeds among diverse taxa has led to the global growth of seed banks to over 1,750 currently, and the conservation of over 50,000 species. Not all plants produce seeds or seeds that survive freezer conditions. Predictive models provide insight into the extent of taxa needing alternative strategies and an initial list of such species is available. These “exceptional” species require cryobiotechnologies (cryogenic storage in liquid nitrogen and in vitro technologies), which provide effective, long‐term ex situ conservation for a wide variety of tissues beyond seeds. The application of cryobiotechnologies increases the potential for conserving all plant biodiversity. Restoration of plant biodiversity into the future will require institutional collaborations among living collections, seed banks, and cryobanks to ensure technology transfer, information gathering and sharing, and capacity building in centers of biodiversity.
Millions of people, particularly in developing nations, depend on collecting exudates from the wild as a means of their livelihood. However, the use and production of exudates have declined in the last 100 years mainly due to the availability of superior and cheaper synthetic alternatives as well as the unsustainable tapping technique that leads to the death of the tapped plant individuals. In this contribution, we present the status of exudates-producing plant species in Southeast Asia, including the diversity, distribution, use, conservation status, protection, and ex situ conservation. The results of the present study can be used as a baseline for evolving a conservation strategy and action plant for exudates-producing plant species in Southeast Asia. Furthermore, the results can also be used to support the sustainable utilization of exudates-producing plant species in the region.
In addition to in-situ conservation activities related to endangered taxa, it has a great importance to provide ex-situ conservation activities to increase awareness and to determine their usage areas by examining reproduction methods. In the line with, the aim of the study is to prevent the extinction of some endangered endemic taxa in Ayaş, Beypazarı and Nallıhan regions of Ankara province in Türkiye, to evaluate the taxa within the framework of sustainable conservation and use principles by raising awareness, and thus to protect the biodiversity in the region. For these purposes, during their vegetation periods throughout the years 2020-2022, some observations were recorded in landscape point of view from the habitats of 9 Critically Endangered (CR) and 3 Vulnerable (VU) taxa. Moreover, all the taxa are photographed and have been archived in the Collections of National Botanical Garden of Türkiye (NBGT) as National Herbarium (TC), DNA and Tissue Banks. Also, samples have been kept in 70% EtOH for Microscobic Slide Collection of NBGT. In addition, the possibilities of using taxa in the landscape area were evaluated. The results of the habitat observations for years serve as a basis for future conservation and landscape gain efforts for the taxa.
Premise:
The effective ex situ conservation of exceptional plants, whether in living collections or cryo-collections, requires more resources than the conservation of other species. Because of their expertise with rare plants, botanical gardens are well positioned to lead this effort, but a well-developed strategy requires a clear understanding of the resources needed.
Methods:
Grant funding was obtained from the Institute of Museum and Library Services to support a three-year project on cryobanking, and to provide smaller grants to 10 other botanical gardens for one-year projects on either (1) seed behavior studies or (2) the development of protocols for in vitro propagation or cryopreservation.
Results:
Nine of the partner gardens worked on 19 species (one was unable to continue due to the COVID-19 pandemic), while the larger project focused on 14 species. A point system was developed for tasks accomplished, and the average costs per point of the larger and smaller projects were similar. Labor accounted for half the costs. Projects focused on species in the Asteraceae and Orchidaceae had lower costs per point than other species.
Discussion:
Both large and small projects can contribute to a strategy for exceptional plant conservation for similar costs. Prioritizing species with lower costs could help advance the field while allowing time for work on more difficult species to develop.
This article reviews the status of plant diversity in the Anthropocene. It considers what is known about the number of species in different plant groups and the geographical distribution of plant diversity. Plants are vitally important in the provision of ecosystem goods and services, and their major values are summarized. As with other components of biodiversity, plant species are threatened by human activities. Knowledge of threatened plant species is discussed. Two major international Conventions aim to protect threatened plant species. One of these, the Convention on Biological Diversity, has agreed targets for plant conservation. Progress in the conservation of plant diversity is briefly reviewed against these targets.
Traditionally, approaches for plant genetic resources conservation have been largely determined by the availability of storage methods. In most cases, genetic resources have been stored as dried seeds at low temperature (for orthodox seed producing species) or as whole plants in field genebanks or botanical gardens (for vegetatively propagated material or species with non-orthodox seed storage behaviour). Additional techniques have recently been developed, including in vitro conservation and pollen storage. Conservation of genetic resources in situ, i.e. in their natural habitat or in farmers' fields, where they have developed distinctive characteristics, is of major importance for maintaining diversity at the ecosystem level (e.g. forest genetic resources) and for cultivated crops and their wild relatives in the centers of diversity.
The different conservation methods complement each other and should be employed in combination, depending on the biological nature of the species, the infrastructural, technical, administrative and policy conditions at the conservation site while considering the needs and requirements of all stakeholders. Whereas genebanks usually aim at the conservation of genetic diversity within cultivated species and their wild relatives and often specialize in a limited number of species or genepools, botanical gardens usually focus on genetic diversity at the species level and maintain large numbers of different (wild) species with little attention being paid to intra-specific diversity. Therefore, botanical gardens and genebanks are complementary for the conservation of plant genetic resources. Consequently, more adequate coordination and cooperation will be beneficial for safeguarding global plant genetic resources for present and future generations.
(This paper is based on a presentation made at the Fifth International Botanic Gardens Conservation Congress; 14 - 18 September, Kirstenbosch, Cape Town, South Africa)
Botanic gardens and arboreta offer the opportunity to conserve and manage a wide range of plant diversity ex situ, and in situ in the broader landscape. The rationale that botanic gardens have a major role to play in preventing plant species extinctions is based on the assumptions that (1) there is no technical reason why any plant species should become extinct, and (2) that, as a professional community, botanic gardens possess a unique set of skills that encompass finding, identifying, collecting, conserving and growing plant diversity across the entire taxonomic spectrum. Botanic Gardens Conservation International (BGCI) is the pivotal centre of a global network of c. 2,600 botanic gardens and arboreta, which includes living collections representing at least one-third of known plant diversity; world class seed banks, glasshouses and tissue culture infrastructures; and technical knowledge networks covering all aspects of plant conservation. Following the example of the crop conservation community, BGCI is promoting the concept of a cost-effective, rational,botanic garden-centred Global System for the conservation and management of plant diversity.This system will aim to collect, conserve, characterise and cultivate samples from all of the world’s rare and threatened plants as an insurance policy against their extinction in the wild and as a source of plant material for human innovation, adaptation and resilience.
The wild relatives of domesticated crops possess genetic diversity useful for developing more productive, nutritious and resilient crop varieties. However, their conservation status and availability for utilization are a concern, and have not been quantified globally. Here, we model the global distribution of 1,076 taxa related to 81 crops, using occurrence information collected from biodiversity, herbarium and gene bank databases. We compare the potential geographic and ecological diversity encompassed in these distributions with that currently accessible in gene banks, as a means to estimate the comprehensiveness of the conservation of genetic diversity. Our results indicate that the diversity of crop wild relatives is poorly represented in gene banks. For 313 (29.1% of total) taxa associated with 63 crops, no germplasm accessions exist, and a further 257 (23.9%) are represented by fewer than ten accessions. Over 70% of taxa are identified as high priority for further collecting in order to improve their representation in gene banks, and over 95% are insufficiently represented in regard to the full range of geographic and ecological variation in their native distributions. The most critical collecting gaps occur in the Mediterranean and the Near East, western and southern Europe, Southeast and East Asia, and South America. We conclude that a systematic effort is needed to improve the conservation and availability of crop wild relatives for use in plant breeding.
Botanic gardens have been exchanging seeds through seed catalogues for centuries. In many gardens, these catalogues remain an important source of plant material. Living collections have become more relevant for genetic analysis and derived research, since genomics of non-model organisms heavily rely on living material. The range of species that is made available annually on all seed lists combined, provides an unsurpassed source of instantly accessible plant material for research collections. Still, the Index Seminum has received criticism in the past few decades. The current exchange model dictates that associated data is manually entered into each database. The amount of time involved and the human errors occurring in this process are difficult to justify when the data was initially produced as a report from another database. The authors propose that an online marketplace for seed exchange should be established, with enhanced search possibilities and downloadable accession data in a standardised format. Such online service should preferably be supervised and coordinated by Botanic Gardens Conservation International (BGCI). This manuscript is the outcome of a workshop on July 9th, 2015, at the European botanic gardens congress “Eurogard VII” in Paris, where the first two authors invited members of the botanic garden community to discuss how the anachronistic Index Seminum can be transformed into an improved and modern tool for seed exchange.
Reintroductions and associated methods have been recommended as techniques for mitigating or redressing threatened plant species declines for several decades. Their use continues to increase as an option for overcoming problems associated with habitat loss, habitat fragmentation, and reproductive isolation (Quinn et al. 1994). However, these approaches have been criticized for the lack of monitoring and central recording, inappropriateness of the action due to genetic considerations, a lack of demographic knowledge of the donor populations, and inadequate information on the species’ habitat needs (Pearman and Walker 2004).
In recent decades, changes that human activities have wrought in Earth’s life support system have worried many people. The human population has doubled in the past 40 years and is projected to increase by the same amount again in the next 40. The expansion of infrastructure and agriculture necessitated by this population growth has quickened the pace of land transformation and degradation. We estimate that humans have modified >50% of Earth’s land surface. The current rate of land transformation, particularly of agricultural land, is unsustainable. We need a lively public discussion of the problems resulting from population pressures and the resulting land degradation.
*Email:
Manuscript received 14 Feb. 2012; accepted 16 Aug. 2012
DOI: 10.1130/GSAT151A.1
A study was designed to develop a routine method for long-term preservation of hardy fruit buds in liquid nitrogen. In hardy shoots of apple ( Malus domestica Borkh.) prefrozen at the temperatures ranging from −30 to −50°C, little or no injury was observed in the leaf buds and cortex after immersion in liquid nitrogen with subsequent slow rewarming at 0°C. When leaf buds from apple shoots immersed in liquid nitrogen for 2 hours after prefreezing to −40° were grafted on the two-year-old seedlings, most of the buds developed normally and continued their shoot growth. Recently, it was confirmed that apple leaf buds immersed in liquid nitrogen for 23 months still remained alive. This prefreezing method was successfully applied to leaf buds of other hardy fruit trees, such as gooseberry, currant, raspberry, and pear.
The in vitro bryophyte collection of the Institute of Biology Bucharest represents the first initiative at national level for bryophyte ex-situ conservation using biotechnological techniques. Micro-propagation and medium-term storage protocols have been developed for 25 bryophyte species of both liverworts and mosses. The collection serves for conservation as well as for research and biotechnological purposes.
The International Treaty on Plant Genetic Resources for Food and Agriculture entered into force in 2004 and is an important instrument by which plant breeders can access crop genetic diversity on the basis of multilateral “facilitated access”. To test how well access works, we sent seed requests to 121 countries that are Contracting Parties to the Treaty. Seeds were received from 44 countries, 54 countries did not respond, while for 23 countries contacts stopped for various reasons: loss of communication, the accessions we requested did not exist or were not in the multilateral system, or conditions or standard material transfer agreements were different from those specified in the Treaty. It is concluded that after nearly 10 years, “facilitated access” is not straightforward.
Background and Aims Quercus species are often considered ‘foundation’ components of several temperate and/or subtropical forest ecosystems. However,
the populations of some species are declining and there is considerable urgency to develop ex situ conservation strategies. In this study, the storage physiology of seeds within Quercus was explored in order to determine factors that affect survival during cryopreservation and to provide a quantitative assessment
of seed recalcitrance to support future studies of this complex trait.
The recent acceleration of actions to conserve plant species using ex situ and in situ strategies has revealed the need to understand how these two approaches might be better developed and integrated in their application to tree species. Here we review some of the recent successes relating mainly to tree seed biology that have resulted in the development and application of innovative actions across five areas: (1) the expansion of living collections to conserve threatened tree species in sufficient numbers to ensure a broad genetic diversity in their progeny; (2) the generation of viability constants to enable estimates to be made of storage longevity of tree seeds in the dry state; (3) improvement in the diagnosis of tree seed storage behaviour through the development of predictive models, reliable prognoses of desiccation tolerance and use of botanical information systems, such as GIS, to correlate information on species distribution and their physiological characteristics; (4) advances in storage preservation biotechnology to enhance the future application of cryopreservation procedures to recalcitrant species in biodiversity hotspots where many are under threat of extinction; (5) integration of ex situ and in situ conservation approaches to ensure that best practice in horticultural and forestry are combined to maintain or enhance genetic diversity, especially in high value species and those with small and vulnerable populations. These actions can lead to greater impact if supported by greater efforts to create seed banks and to collate databases world-wide so that data, knowledge and collections are more available to the scientific, forestry and NGO communities. Throughout this review we have used examples from the mega-biodiversity countries of Brazil and China, as a way of illustrating wider principles that can be applied in many countries. Future development of current research approaches, the adherence to conservation policy and the expanding needs for education are also considered briefly.
Like all other plants, bryophytes suffer from various threats in a rapidly changing environment. Compared with vascular
plants, they have received significantly less conservation attention, and the issues around their conservation have been a
subject of study for little more than 30 years. Very few documented actions have been taken to halt the decline in bryophyte
diversity (including genetic, species and habitat conservation). The ex situ approach is one of the most recently developed
methods in the conservation initiative but it is rarely applied to bryophyte conservation. Lately, significant advances have
been made in in vitro bryophyte biotechnology as a component of ex situ propagation. In this article, we discuss the current
state of knowledge, associated problems and achievements, with reference to the ex situ bryophyte collection of the Bryophyte
Biology Group in Belgrade.
Phenological monitoring at the Royal Botanic Garden Edinburgh (RBGE) began in the mid 19th century, and is now being developed as a number of projects. In view of the wide range of plantclimate interactions, it is recommended that projects are designed with clear and limited objectives, and are then conducted consistently and to a high standard over a long period. The projects at RBGE are outlined, and the suitability of botanic gardens in general for phenology is discussed. A distinction is drawn between 'organism phenology' and 'population phenology', and also between 'extensive' and 'intensive' approaches to project design. The variety of possible projects is illustrated by a number of completed and on-going projects in the UK, USA and northern Eurasia. It is suggested that botanic gardens can enhance their service to society by becoming phenological monitoring stations.
It is concluded from a review of the literature that plant cell culture itself generates genetic variability (somaclonal variation). Extensive examples are discussed of such variation in culture subclones and in regenerated plants (somaclones). A number of possible mechanisms for the origin of this phenomenon are considered. It is argued that this variation already is proving to be of significance for plant improvement. In particular the phenomenon may be employed to enhance the exchange required in sexual hybrids for the introgression of desirable alien genes into a crop species. It may also be used to generate variants of a commercial cultivar in high frequency without hybridizing to other genotypes.
The high mountain pottioid moss Molendoa hornschuchiana (Hook) Lindb. ex Limpr. is a very rare and critically endangered bryophyte species in Europe in need for ex situ conservation. A 25-year-old herbarium sample was used to revive and propagate this species for further reintroduction and introduction to potential natural habitats. The reviving of “dead” herbarium specimen was achieved by disposing of axenical organisms as well as adjusting condition for developing secondary protonema, bud inductions, and optimization of gametophyte propagation in vitro condition.
The influence of exogenously added growth regulators on the morphogenesis of this species was studied. The plants were cultured in the two basic types of media, viz., BCD and half-strength Murashige and Skoog (MS) supplemented with different concentrations (0.01–0.3 μM) of indole-3-butyric acid (IBA) and N 6 -benzyladenine (BA) under a 16-h photoperiod. The influence of growth regulators on gametophores multiplication in vitro as well as on protonemal diameter was recorded. Well-developed gametophores were obtained on BCD medium, whereas on half-strength MS medium, secondary protonema was produced, both on hormone-free and supplemented substrate exclusively. Based on multiplication index in vitro, maximum development of gametophores was realized on BCD medium supplemented with 0.3 μM IBA and 0.1 μM BA. However, the widest diameter of secondary protonema was obtained on BCD medium enriched with low concentration of both BA (0.01 and 0.03 μM) and constant concentration of IBA (0.03 μM). Chemical names used: indole-3-butyric acid (IBA), N 6 -benzyladenine (BA), Murashige and Skoog medium (MS).
The moss Entosthodon hungaricus (Boros) Loeske is an European endemic species typical of dry and saline soils extending from the Iberian Peninsula to Aral-Caspian steppes, similarly to some other xerothermic bryophytes. However, the distribution range is fragmented and localities are quite scattered and the species is considered as rare and vulnerable because of its ephemeral characteristics and specialized ecology.
With the aim to develop an active protection plan for this species, the ex situ conservation requirements of E. hungaricus were developed. The axenic culture in in vitro conditions were established, and the optimal growth parameters were adjusted to achieve fully developed gametophytes ready to be reintroduced to its native range and other potentially native areas, where this species was once reported but has not been collected in recent times, suggesting its local extinction (i.e. some areas in Vojvodina, N. Serbia). Starting materials were derived from recent herbarium specimens and from fresh materials collected from Hungarian populations. Several means for sterilization of stating material and growing nutritive media were assayed in different regimes of light and temperature.
Here we describe the conditions to achieve full plant development and for its micropropagation. Such materials are adequate for ex situ conservation purposes and for experimental introductions in native and potentially native areas. The first axenical culture of E. hungaricus was successfully established, and the first in vitro micropropagation of this rare and endangered species was achieved. Our study contributes to the conservation biology as well as for the potential use of this moss species in biotechnological research.
The brown alga Ectocarpus has recently become the first fully sequenced multicellular alga and is an important biological model. Due to the large and growing number of Ectocarpus strains isolated and maintained by the research community, including increasing numbers of mutants, there is an urgent need for developing reliable, cost-effective long-term maintenance techniques. We report here that cryopreservation constitutes an attractive option in this respect, using a simple two-step protocol employing combined DMSO 10% (v/v) and sorbitol 9% (w/v) as cryoprotectants. This model organism appears to be remarkably robust and post-cryo recovery has been observed in all strains tested in this study. Cultures can be regenerated by the germination of cryopreserved zooids (spores), or the recovery of vegetative cells. In the latter case, dividing surviving cells may grow into the cell lumen of a neighbouring dead cell, eventually regenerating a phenotypically normal thalloidal structure.
Across the Canadian Arctic Archipelago, widespread ice retreat during the 20th century has sharply accelerated since 2004. In Sverdrup Pass, central Ellesmere Island, rapid glacier retreat is exposing intact plant communities whose radiocarbon dates demonstrate entombment during the Little Ice Age (1550-1850 AD). The exhumed bryophyte assemblages have exceptional structural integrity (i.e., setae, stem structures, leaf hair points) and have remarkable species richness (60 of 144 extant taxa in Sverdrup Pass). Although the populations are often discolored (blackened), some have developed green stem apices or lateral branches suggesting in vivo regrowth. To test their biological viability, Little Ice Age populations emerging from the ice margin were collected for in vitro growth experiments. Our results include a unique successful regeneration of subglacial bryophytes following 400 y of ice entombment. This finding demonstrates the totipotent capacity of bryophytes, the ability of a cell to dedifferentiate into a meristematic state (analogous to stem cells) and develop a new plant. In polar ecosystems, regrowth of bryophyte tissue buried by ice for 400 y significantly expands our understanding of their role in recolonization of polar landscapes (past or present). Regeneration of subglacial bryophytes broadens the concept of Ice Age refugia, traditionally confined to survival of land plants to sites above and beyond glacier margins. Our results emphasize the unrecognized resilience of bryophytes, which are commonly overlooked vis-a-vis their contribution to the establishment, colonization, and maintenance of polar terrestrial ecosystems.
The use of in vitro techniques for conservation has been rising steadily since their inclusion in The Convention on Biological Diversity and
The Global Strategy for Plant Conservation. Unfortunately, bryophytes are often overlooked in conservation initiatives, but
they are important in a number of large-scale ecosystem processes, i.e. nutrient, water and carbon cycling. There is a long history of the use of tissue culture in cultivating bryophytes, and many
species respond well to in vitro techniques. For 6yr (2000–2006), The Royal Botanic Gardens, Kew and the UK statutory conservation agencies supported a project
for the ex situ conservation of bryophytes. Living and cryopreserved collections of UK threatened species were successfully established and
the cryopreserved collection continues to be maintained. Other in vitro conservation collections are maintained over Europe, at botanic gardens, museums and by individual university researchers,
but there is no coherent European collection of bryophytes for conservation, or standardisation of techniques. A major issue
for many in vitro collections is the maintenance of within species genetic diversity. Such diversity is considered to be important, as it is
the basis by which populations of species can adapt to new conditions and evolve. We are proposing to establish a European
network for in vitro conservation of bryophytes. We envisage that this will include living collections, cryopreserved collections and spore collections.
Conservation of genetic diversity would be a priority and the collections would provide a valuable resource for conservation
initiatives and support research into rare and threatened species.
KeywordsBryophyte–Tissue culture–Cryopreservation–
Ex situ conservation–European collections
Preservation of clonal genotypes of most fruit crops requires vegetative propagation, so cryopreservation is an ideal method
for long-term germplasm storage (Engelmann 2004). Some of the first and most successful experiments with the cryopreservation
of apical meristems began with a temperate berry crop, strawberry (Kartha et al. 1980; Sakai et al. 1978). These initial studies
showed that slow cooling of <1 mm shoot tips in cryoprotectant solutions was a feasible option for long-term storage of clonally
propagated plants. During the last two decades, cryopreservation methods were developed for many species of fruit crops (Reed
2001).
Seeds are categorized into two main groups according to their response to desiccation and their storage physiology: orthodox (desiccation-tolerant) and recalcitrant (desiccation-sensitive) seeds (Roberts 1973). A third category of seeds are those that are relatively desiccation tolerant but do not withstand desiccation down to water contents as low as those tolerated by orthodox seeds. These seeds are freezing sensitive and are referred to as intermediate seeds (Ellis et al. 1990, 1991).
Seed is the most preferred plant propagule for ex situ germplasm conservation due to low storage cost, ease of seed handling and regeneration of whole plants from genetically diverse materials (Chin 1994; Pritchard 1995). While orthodox seeds are acquiescent to storage under conventional gene bank conditions for centuries, (i.e. 3–7% seed water content at –20°C ) (FAO/IPGRI 1994), cryopreservation is the only available option for longterm storage of non-orthodox seeds. However, in circumstances where storage of the whole seed of a non-orthodox species is constrained by desiccation and freezing sensitivity on one hand, and by its relatively large seeds on the other, excised embryos and embryonic axes are an alternative option. In other cases where viability of lipid-rich orthodox seeds under conventional storage conditions is drastically reduced due to the thin seed coat coupled with lipid peroxidation, as in the case of peanuts (Arachis hypogaea), germplasm curators can resort to cryopreservation of the excised embryonic axes (Gagliardi et al. 2002). Several studies on recalcitrant and intermediate species empirically determined that excised embryos and embryonic axes (in most cases) are more tolerant to desiccation and subsequent cryoexposure than whole seeds (e.g. Bajaj 1984; Radhamani and Chandel 1992; Normah et al. 1994; Makeen et al. 2005)
Controlled rate cooling is based on osmotic regulation of cell contents and freeze-induced dehydration. The samples are pretreated in cryoprotectant solutions and cooled at a standard rate to an intermediate temperature such as –35°C or –40°C, with ice nucleation initiated at about –9°C. At the freezing point of the cryoprotectant solution, ice nucleation is initiated, and ice forms in the cryoprotectant solution and the intercellular spaces. The cytoplasm remains unfrozen due to solute concentration and the cell wall protects the cell membrane from damaging ice crystals. As the temperature is further decreased to –35°C or –40°C, the extracellular solution becomes increasingly icy and the intracellular solutes become highly concentrated. The plant cells lose water to the exterior ice and the cytoplasm is further concentrated. The intracellular freezable water is safely reduced before samples are plunged into liquid nitrogen (LN). If the cells are optimally dehydrated, the cytoplasm vitrifies on contact with LN. If the samples are under dehydrated, leaving freezable water in the cytoplasm, ice will form. If over dehydrated, the cells may die from desiccation.
Controlled rate cooling is very efficient for storing suspension and callus cultures, embryogenic cultures, and in-vitro shoot tips from temperate and subtropical plants. The advantages of controlled rate cooling include the use of standardized procedures, programmed cooling rates, and large batch sizes, and the effective use of technician time.
Premise of the study:
Fern spores are unicellular and haploid, making them a potential model system to study factors that regulate lifespan and mechanisms of aging. Aging rates of nongreen spores were measured to compare longevity characteristics among diverse fern species and test for orthodox response to storage temperature and moisture.
Methods:
Aging of spores from 10 fern species was quantified by changes in germination and growth parameters. Storage temperature ranged from ambient room to -196°C (liquid nitrogen); spores were dried to ambient relative humidity (RH) or using silica gel.
Key results:
Survival of spores varied under ambient storage conditions, with one species dying within a year and two species having greater than 50% survival after 3 years. Few changes in germination or growth were observed in spores stored at either -80°C or -196°C over the same 3-yr study period. Spores stored at -25°C aged anomalously quickly, especially those dried to ambient RH or subjected to repeated freeze-thaw cycles.
Conclusions:
Spore longevity is comparable to orthodox seed longevity under ambient storage conditions, with wide variation among species and shelflife extended by drying or cooling. However, faster aging during freezer storage may indicate a similar syndrome of damage experienced by seeds categorized as "intermediate". The damage is avoided by storage at -80°C or liquid nitrogen temperatures, making cryoconservation an effective and broadly applicable tool to extend fern spore longevity. The study demonstrates that spore banks are a feasible approach for ex situ conservation of this important plant group.
Green spores of ferns lose viability quickly, and need specialized treatment for long-term conservation in germplasm banks. Dry storage at different temperatures was studied in green spores of Osmunda regalis and Equisetum ramosissimum. Changes in germination percentage, time to 50 percent of maximum germination (T50) and tendency for normal growth of the gametophyte were assayed during 24 months of storage. Spores stored at 25 degree C died within 1 month. Spores stored at 4 degree C maintained high viability for about 3 months, and then aging was evident by a decrease of final germination percentage, an increase in T50, and abnormal development of the gametophyte. Germination of spores stored at -25 degree C was highly variable during the storage period. Spores cryopreserved at -80 degree C and -196 degree C maintained high viability, rapid germination and normal growth throughout the study period. Cryopreservation of green spores is a feasible method to preserve viability and ensure normal gametophyte development for several years.
Bryophytes are a non-monophyletic group of three major lineages (liverworts, hornworts, and mosses) that descend from the earliest branching events in the phylogeny of land plants. We postulate that desiccation tolerance is a primitive trait, thus mechanisms by which the first land plants achieved tolerance may be reflected in how extant desiccation-tolerant bryophytes survive drying. Evidence is consistent with extant bryophytes employing a tolerance strategy of constitutive cellular protection coupled with induction of a recovery/repair mechanism upon rehydration. Cellular structures appear intact in the desiccated state but are disrupted by rapid uptake of water upon rehydration, but cellular integrity is rapidly regained. The photosynthetic machinery appears to be protected such that photosynthetic activity recovers quickly. Gene expression responds following rehydration and not during drying. Gene expression is translationally controlled and results in the synthesis of a number of proteins, collectively called rehydrins. Some prominent rehydrins are similar to Late Embryogenesis Abundant (LEA) proteins, classically ascribed a protection function during desiccation. The role of LEA proteins in a rehydrating system is unknown but data indicates a function in stabilization and reconstitution of membranes. Phylogenetic studies using a Tortula ruralis LEA-like rehydrin led to a re-examination of the evolution of desiccation tolerance. A new phylogenetic analysis suggests that: (i) the basic mechanisms of tolerance seen in modern day bryophytes have changed little from the earliest manifestations of desiccation tolerance in land plants, and (ii) vegetative desiccation tolerance in the early land plants may have evolved from a mechanism present first in spores.
Microalgae are one of the most biologically important elements of worldwide ecology and could be the source of diverse new products and medicines. COBRA (The COnservation of a vital european scientific and Biotechnological Resource: microAlgae and cyanobacteria) is the acronym for a European Union, RTD Infrastructures project (Contract No. QLRI-CT-2001-01645). This project is in the process of developing a European Biological Resource Centre based on existing algal culture collections. The COBRA project's central aim is to apply cryopreservation methodologies to microalgae and cyanobacteria, organisms that, to date, have proved difficult to conserve using cryogenic methods. In addition, molecular and biochemical stability tests have been developed to ensure that the equivalent strains of microorganisms supplied by the culture collections give high quality and consistent performance. Fundamental and applied knowledge of stress physiology form an essential component of the project and this is being employed to assist the optimisation of methods for preserving a wide range of algal diversity. COBRA's "Resource Centre" utilises Information Technologies (IT) and Knowledge Management practices to assist project coordination, management and information dissemination and facilitate the generation of new knowledge pertaining to algal conservation. This review of the COBRA project will give a summary of current methodologies for cryopreservation of microalgae and procedures adopted within the COBRA project to enhance preservation techniques for this diverse group of organisms.
This volume presents a comprehensive review of reintroduction projects and practices, the circumstances of their successes or failures, lessons learned, and the potential role for reintroductions in preserving species threatened by climate change. Contributors examine current plant reintroduction practices, from selecting appropriate source material and recipient sites to assessing population demography. The findings culminate in a set of Best Reintroduction Practice Guidelines, included in an appendix to the book. These guidelines cover stages from planning and implementation to long-term monitoring, and offer not only recommended actions but also checklists of questions to consider that are applicable to projects around the world. Plant Reintroduction in a Changing Climate is a comprehensive and accessible reference for practitioners to use in planning and executing rare plant reintroductions.
Combining community needs and preferences with dryland plant expertise in order to select suitable native species for large-scale natural capital restoration is the approach that has been successful in the Sahel as part of Africa's Great Green Wall program. In order to increase plant diversity and restore degraded land, we investigated four cross-border regions of Mali, Burkina Faso, and Niger, all located in dryland ecosystems of the Sahel. In 120 beneficiary village communities, with a total population of over 50,000 farmers, including 51% women, participatory diagnostic meetings were conducted, leading to the selection of 193 plant species, most of which were mainly used for food, medicine, fodder, and fuel. Of these, 170 were native and considered suitable for enriching and restoring those village lands. The most environmentally well-adapted and economically relevant species were prioritized, quality seeds were collected, and nursery seedlings produced under technical supervision of villages. From 2013 to 2015, 55 woody and herbaceous species were planted to initiate restoration of 2,235 ha of degraded land. On average, 60% of seedlings survived and grew well in the field after three rainy seasons. Due to its multiple uses, including gum arabic production, Acacia senegal was preferred by local people in most cases, accounting for 30% of seedlings planted. Such promising results, in an effort to restore degraded land for and with the help of thousands of farmers, could not have been achieved without the combination of scientific plant expertise and efficient rural capacity development, underpinned by high levels of community engagement.
Cryopreservation is needed to ensure long-term storage without any genetic drift and contamination. To conserve Ulva species, gametophytic thalli of Ulva prolifera (Chlorophyta) were first cryopreserved at −196 °C using the two-step cooling method with cooling rate of 1 °C min−1 to −40 °C. Three cryoprotectants (dimethyl sulfoxide (DMSO), glycerol, and proline) at five concentrations (5, 10, 15, 20, and 25 %) and mixed cryoprotectants were assessed to determine the optimal solution. The mixture of cryoprotectants with the highest viability was 10 % glycerol in combination with 5 % DMSO and 5 % proline, with a viability of 89.2 %. The cryoprotectant treated singly with the highest viability was 20 % glycerol with a viability of 91.6 %. However, there was no statistically significant difference between 20 % glycerol and the mixed treatment. An evaluation of long-term storage in liquid nitrogen showed very high viability of 91.2–92.1 % with no statistically significant effect from storage time up to 120 days. The specimens developed normally in culture after cryopreservation for 120 days with the rate of gametogenesis reaching 95.7 % on the fourth day of culture. The released gametes developed normally into gametothalli. In conclusion, the use of glycerol as a cryoprotectant was very effective for cryopreservation of the gametothalli of U. prolifera. With 20 % glycerol, viability was very high as 91.6 %. The results suggest that gametothalli of U. prolifera can be cryopreserved completely by a treatment with 15–25 % glycerol singly as well as by a mixture of cryoprotectants with usual efficiency as a cryoprotectant.
Thirty-three species of ferns with nonchlorophyllous spores and five species with chlorophyllous spores were studied in regard to their ability to survive exposure to liquid nitrogen (LN). Air dried spores showed no inhibition of germination after LN exposure when planted on soil or growth medium. Spores of three species that were stored for 75 months either at 4°C, -20°C, or in LN showed no decrease in viability over that time, and spores of four other species were maintained successfully for 52 months in LN. Fresh chlorophyllous spores that were air dried, dried over silica gel, or prepared with the encapsulation dehydration procedure also showed good survival through desiccation and LN exposure. Spores of Osmunda regalis germinated well after 18 months of LN storage. These results indicate that both nonchlorophyllous and chlorophyllous spores are candidates for long-term germplasm storage at low temperatures, including storage in LN.
The single germ tubes produced by the spores of Ephemerum comprise highly chlorophyllose cells divided by transverse cross-walls. All subsequent cross-walls are oblique and thus the protonemata of Ephemerum would appear to be almost entirely caulonemal. Initially, the protonema comprises a loose mat of irregularly-branched filaments. Subsequently, leading filaments around the margins produce upright determinate branching systems which give the protonema of Ephemerum its distinctive appearance. The ultimate ramifications of these systems terminate in sharp points with thickened walls whilst their regular branching is predetermined by zig-zag development of the young side branches. Unlike all the other parts of the protonema, the determinate branching system fails to regenerate when transplanted onto new medium. Whereas cytokinins stimulate bud formation and cause the formation of highly attenuate filaments, abscisic acid induces the production of chains of mucilage-invested spherical brood cells, a phenomenon also seen in ageing cultures. These are desiccation resistant and germinate rapidly on new medium. Nutrient-free agar has little effect on protonemal morphogenesis.
To improve storage of green spores by liquid nitrogen (LN), we studied the influence of spore maturity and desiccation process on storage of Osmunda japonica spores. Germination percentages of mature spores after drying for 12 h or equilibrating at 10–55 % relative humidity did not significantly differ from those of fresh spores. So we established that the critical water contents of mature spores were about 6.6 and 4.8 % by silica gel and equilibrating with saturated salt solutions, respectively. Germination percentages of post-mature spores after desication were lower than those of fresh spores. The viability of mature spores was always higher than the viability of post-mature spores following varying periods of storage in LN. The germination percentages of mature spores stored in LN after silica gel desication were comparable to those of mature spores desiccated with saturated salt solution, while desiccation with silica gel led to a marked decrease in germination percentages of post-mature spores during subsequent storage. In general, the level of maturity and desiccation process can have an impact on storage of green spores by LN storage. Mature spores can tolerate desiccation and freezing independent of the desication process. However, slow removal of intracellular water led to less damage and provided more protection to post-mature spores during storage. In addition, reduction in water contents of spores to approx. their critical levels can achieve the best results for long-term storage of spores.
Two methods of protecting fern gametophyte tissues through exposure to liquid nitrogen (LN) were examined. In vitro grown gametophytic tissues from six fern species were exposed to LN after open drying or after encapsulation dehydration, with and without preculture on abscisic acid (ABA). Open drying itself decreased survival with little further effect from LN exposure, although survival was somewhat improved by preculture on ABA. In contrast, encapsulated tissues survived drying and LN exposure at rates comparable to controls (86-100%) irrespective of ABA preculture. Sucrose pretreatment of the encapsulated tissues was important for their subsequent survival through these procedures. Tissues prepared by encapsulation dehydration were successfully regrown after 3.5 years in LN storage. Thus, cryopreservation appears to be a technique which could be used for the stable preservation of in vitro cultures of fern gametophytes and for the long-term storage of rare or endangered germplasm of ferns.
Seaweeds inhabiting the upper intertidal zone are subjected to temperature, light, and water stresses and vertical distribution has been linked to environmental tolerance. Previous studies have also attributed successful recovery from freezing stress in intertidal seaweeds to desiccation tolerance. Porphyra umbilicalis Kützing is an aseasonal red alga inhabiting the mid to upper intertidal zone in temperate and subarctic regions of the North Atlantic. It is a member of the economically important group of foliose Bangiales, and has been documented to only reproduce asexually via neutral spores in the Northwest Atlantic. The goal of this study was to assess the effects of freezing on the viability of small blades of P. umbilicalis. Cultured blades of P. umbilicalis (4.8 ± 0.22 mg) were air dried to 5% or 30% absolute water content (AWC) and frozen for 1, 3, 6, or 12 months at − 80 °C or − 20 °C. Following freezing, blades were rehydrated and the growth rate of each blade was measured weekly for 4 weeks. Photosynthetic efficiency of photosystem II (Fv/Fm) was assessed for each blade 3 h and 4 weeks post-rehydration. Overall, there was 100% blade survival and all blades continued to grow after rehydration. Although the conditions under which the blades were frozen did have statistically significant effects on post-rehydration growth rate and Fv/Fm, in general the differences were quite small. Post-rehydration growth rates ranged from 7.06 to 8.03 ± 0.16% day− 1. AWC had an effect on post-rehydration growth rates for blades frozen at − 80 °C, but not blades frozen at − 20 °C. The length of freezing had a somewhat greater effect on blades with 5% AWC than blades with 30% AWC. Growth rates peaked two weeks post-rehydration followed by a small decline in weeks 3 and 4. Fv/Fm values following freezing were generally similar to those recorded in previous studies on non-frozen blades; however, blades frozen for 6 months performed better than blades frozen for 12 months. Overall, these results indicate that short- and long-term freezing have little physiological effect on blades of P. umbilicalis. Therefore, freezing may be a viable method for preservation of P. umbilicalis for aquaculture.
Aim
Correlative models that forecast extinction risk from climate change and invasion risks following species introductions, depend on the assumption that species' current distributions reflect their climate tolerances (‘climatic equilibrium’). This assumption has rarely been tested with independent distribution data, and studies that have done so have focused on species that are widespread or weedy in their native range. We use independent data to test climatic equilibrium for a broadly representative group of species, and ask whether there are any general indicators that can be used to identify when equilibrium occurs.
Location
E urope and contiguous USA .
Methods
We contrasted the climate conditions occupied by 51 plant species in their native ( E uropean) and naturalized ( USA ) distributions by applying kernel smoothers to species' occurrence densities. We asked whether species had naturalized in climate conditions that differ from their native ranges, suggesting climatic disequilibrium in the native range, and whether characteristics of species' native distributions correspond with climatic equilibrium.
Results
A large proportion of species' naturalized distributions occurred outside the climatic conditions occupied in their native ranges: for 22 species, the majority of their naturalized ranges fell outside their native climate conditions. Our analyses revealed large areas in E urope that species do not occupy, but which match climatic conditions occupied in the USA , suggesting a high degree of climatic disequilibrium in the native range. Disequilibrium was most severe for species with native ranges that are small and occupy a narrow range of climatic conditions.
Main conclusions
Our results demonstrate that the direct effects of climate on species distributions have been widely overestimated, and that previous large‐scale validations of the equilibrium assumption using species' native and naturalized distributions are not generally applicable. Non‐climatic range limitations are likely to be the norm, rather than the exception, and pose added risks for species under climate change.
The nucellar cells of navel orange(Citrus sinensis Osb. var. brasiliensis Tanaka) were successfully cryopreserved by vitrification. In this method, cells were sufficiently dehydrated with highly concentrated cryoprotective solution(PVS2) prior to direct plunge in liquid nitrogen. The PVS2 contains(w/v) 30% glycerol, 15% ethylene glycol and 15% DMSO in Murashige-Tucker medium(MT) containing 0.15 M sucrose. Cells were treated with 60% PVS2 at 25°C for 5 min and then chilled PVS2 at 0°C for 3 min. The cell suspension of about 0.1 ml was loaded in a 0.5 ml transparent plastic straw and directly plunged in liquid nitrogen for 30 min. After rapid warming, the cell suspension was expelled in 2 ml of MT medium containing 1.2 M sucrose. The average rate of survival was about 80%. The vitrified cells regenerated plantlets. This method is very simple and the time required for cryopreservation is only about 10 min.
The current distributions of species are often assumed to correspond with the total set of environmental conditions under which species can persist. When this assumption is incorrect, extinction risk estimated from species distribution models can be misleading. The degree to which species can tolerate or even thrive under conditions found beyond their current distributions alters extinction risks, time lags in realizing those risks, and the usefulness of alternative management strategies. To inform these issues, we propose a conceptual framework within which empirical data could be used to generate hypotheses regarding the realized, fundamental, and 'tolerance' niche of species. Although these niche components have rarely been characterized over geographic scales, we suggest that this could be done for many plant species by comparing native, naturalized, and horticultural distributions.
Maintaining numbers of aseptic cultures of bryophytes by serial transfer not only consumes supplies and labor but can result in loss of vigor or the appearance of other abnormalities in long-term cultures. Cryopreservation of cultures solves the problems associated with long-term culture, and is of particular importance for cultures or mutant cell lines incapable of making spores or where spores are short-lived. Dramatic simplification of a published protocol for moss cryopreservation is accomplished by preconditioning cultures for 3-4 days in medium supplemented with 10-5 M ABA and 100 mM proline. This proline/ABA protocol can be successfully used with Ceratodon purpureus, Funaria hygrometrica, Physcomitrella patens, and two species of Sphagnum; cryopreserved cultures remain viable for a minimum of one year at -80°C. Experiments demonstrate a differential response by cell types to cryopreservation, as well as differences between species or for particular selected cell lines within a species. Other experiments suggest that successful cryopreservation is not a simple consequence of brood cells in ABA-treated cultures.
The formation of ovoid or spherical brood cells from chloronemal filaments by swelling and symmetrical subapical divisions preceded by phragmosomes, but not preprophase bands, is described in the mosses Physcomitrella patens, Dicranoweisia cirrata, Bryum tenuisetum, Bryum bicolor, and Rhytidiadelphus loreus. This is a widespread phenomenon that occurs in protonemal colonies cultured for long periods of time or allowed to dry out. It involves the redifferentiation of highly polarized chloronemal cells with a net axial array of microtubules into nonpolar cells containing microtubules with no preferred orientation. Brood cells in some species become thick walled and retain their viability for long periods even in a desiccated state. When transferred to new medium they immediately regenerate protonemata from new filaments of no fixed position. Experiments using activated charcoal, which prevents brood cell formation, and transferring protonemata or spores onto old medium in which brood cells had been produced previously, indicate that the redifferentiation process is triggered by substances released into the medium by protonemata. Addition of abscisic acid, an endogenous growth regulator in mosses, causes precocious brood cell formation in young cultures and is very likely the natural compound triggering their development and inducing their tolerance to desiccation. Key words: abscisic acid, activated charcoal, redifferentiation, desiccation tolerance, microtubules, polarity, protonema, regeneration.
Cryopreservation is a practical method for maintaining a broad range of microalgae over long periods of time. The Culture Collection of Algae at the University of Texas at Austin (UTEX) currently successfully maintains over 1300 strains of algae under cryopreservation, although many other strains at UTEX cannot yet be cryopreserved or else survive with very low viability. The ability of a culture of microalgae to survive freezing and thawing is strongly dependent on the conditions under which it is frozen, including the nature and concentration of a cryoprotective agent added to the algal culture prior to freezing. Experiments using the unicellular microalga Chlorococcum texanum Archibald et Bold (UTEX #1788), demonstrate that methanol is transported across the plasma membrane faster than water, while dimethyl sulfoxide is transported more slowly than water. These differences cause very different volumetric excursions of algal cells in response to the added cryoprotective agent. This may help explain why different algae respond differently to methanol and dimethyl sulfoxide, although both are widely used as cryoprotective agents. An often overlooked parameter during cryopreservation of algae is the culture density at the time of freezing. Studies using Chlamydomonas reinhardtii Dangeard (UTEX #89) demonstrate that low culture density during freezing is important for high viability after thawing from liquid nitrogen. The low viability of C. reinhardtii cultures cryopreserved at high cell density is caused by an unidentified water-soluble substance that is released into the medium from cells that are killed during cryopreservation. At sufficiently high concentration the substance is lethal to other cells in the medium when the cryopreserved culture is thawed. The inhibitory substance is small, water soluble and heat-stable, but its release from cells is prevented by heat-denaturing the algal cells prior to their freezing and thawing.
The Center for Plant Conservation (CPC) has created sampling guidelines for the ex situ conservation of rare plant species. These guidelines estimate the number of individuals needed to maximize the genetic diversity of the collection according to population genetic theory. For many clonal plant species, knowledge of the number of unique individuals is not easily discerned and application of these guidelines must be based on molecular genetic data. In this paper, we discuss the steps taken in order to meet CPC guidelines for the conservation of a rare clonal plant, Clematis socialis. Due to limited seed availability, methods were developed for successful in vitro propagation and cryopreservation of C. socialis shoot tips. Inter-simple sequence repeat (ISSR) analysis identified fifteen unique genotypes in the ex situ in vitro collection. One genotype in this collection has been conserved from a population that is now presumed extinct. Although the initial sampling protocol managed to capture considerable genetic diversity, an additional 97 genotypes are needed to meet CPC guidelines. The information and experience gained through the initial C. socialis ex situ conservation efforts form the basis for a strategy to improve ex situ conservation activities for this endangered species. We recommend that additional in vitro collections be made from each of the five extant populations and placed in cryostorage.
The Millennium Ecosystem Assessment estimates that between 60,000 and 100,000 plant species are threatened with extinction–equivalent to around one-quarter of the total number of known plant species. Why should we care? There are a number of reasons. The first is that these plants may be useful to us in unknown ways. Secondly, ecology has taught us that resilience is found in diversity. Thirdly, we should be saving plant species from extinction because we can–there is no technological reason why any plant species should become extinct. Where we can't protect and manage plant diversity in situ, we should be employing ex situ conservation techniques, ranging from seed banks to habitat restoration. The Millennium Ecosystem Assessment describes such interventions as ‘techno-gardening’. This is not an abstract concept–it is already a reality in the majority of man-managed landscapes. In this context the perception of ex situ conservation as simply a back-up strategy for in situ conservation is mistaken. We are all involved in ex situ conservation to some degree, from cultivating our back gardens, to farming, to management of protected areas. Ex situ conservation should be seen as a complementary approach to in situ conservation and on the same spectrum. Kew's Millennium Seed Bank Partnership, comprising more than 120 plant science institutions in 50 countries, epitomizes this philosophy in action. We work actively on every seed collection we bank, finding out how useful it is and how we can grow it to enable human innovation, adaptation and resilience. Challenges remain at the policy level; for example, the need to factor-in the value of natural capital to development decision making, and better defining a role for public-sector science. At the technical level, also, there is much to do. Perhaps the greatest technical challenges relate to the restoration and management of complex, self-sustaining habitats or species assemblages. If we are to techno-garden effectively, in order to maintain ecosystem services and sustain biodiversity, then a multidisciplinary approach will be required. Many plant science institutions have recognized this and are becoming engaged increasingly in restoration activities and in situ management. Ultimately, humanity's ability to innovate and adapt is dependent on our having access to the full range of plant species and the alleles they contain.
Seeds of different species are believed to have characteristic shelf lives, although data confirming this are scarce, and a mechanistic understanding of why this should be remains elusive. We have quantified storage performance of c. 42,000 seed accessions, representing 276 species, within the USDA National Plant Germplasm System (NPGS) collection, as well as a smaller experiment of 207 cultivars from 42 species. Accessions from the NPGS collection were harvested between 1934 and 1975, and had relatively high initial germination percentages that decreased at a variable rate during storage at both 5 and –18°C. Germination time courses, which represent the average performance of the species, were fitted to Avrami kinetics, to calculate the time at which germination characteristically declined to 50% (P50). These P50 values correlated with other longevity surveys reported in the literature for seeds stored under controlled conditions, but there was no correlation among these studies and seed persistence observed in the classic buried seed experiment by Duvel. Some plant families had characteristically short-lived (e.g. Apiaceae and Brassicaceae) or long-lived (e.g. Malvaceae and Chenopodiaceae) seeds. Also, seeds from species that originated from particular localities had characteristically short (e.g. Europe) or long (e.g. South Asia and Australia) shelf lives. However, there appeared to be no correlation between longevity and dry matter reserves, soluble carbohydrates and parameters relating to soil persistence or resource allocation. Although data from this survey support the hypothesis that some species tend to survive longer than others in a genebank environment, there is little information on the attributes of the seed that affect its storage performance.
Gametophytes are more abundant thou sporophytes in wave exposed rocky intertidal populations of Iridaea laminarioides Bory in Central Chile. In this study we experimental tested the differential effects of selected ecological factors on karyologically different life history phases. In the field, gametophytes dominated at higher elevation and during summer; tetrasporophytes were most abundant low in the intertidal and during the fall. Laboratory responses correlated with these patterns. Gametophytes exhibited greater desiccation tolerance than tetrasporophytes. Optimum growth of gametophytes occurred at higher temperatures (20°C) and longer photoperiods (16:8 h LD) than sporophytes (15°C and 12:12 h LD). Grazing preferences changed with the developmental stage of the alga, but all herbivores tested had increased preference for diploid tissues as compared to haploid. Number of spores produced with respect to total plant surface, or total rocky surface, or settlement of spores and their germination rate did not show significant differences between phases but showed great variability in space and time. Spontaneous spore release, however, was always higher in cystocarpic than in tetrasporangial thalli. Such a combination of results suggests that some real ecological differences exist between the two life history phases of I. laminarioides. Such ecological differences permit a prediction of vertical and temporal patterns of distribution for both phases. Horizontal patterns of distribution cannot be explained because the several selection factors probably interact differently in various habitats.
In vitro methods provide opportunities for propagating and preserving endangered plant species when seed-based methods are not adequate.
Such species include those that produce few or no seeds, as well as species with recalcitrant seeds. Tissue culture propagation
methods can be used to produce such plants for reintroduction, research, education, display, and commerce. They can also be
the basis for tissue banking as a way to preserve genetic diversity when seeds cannot be banked. With some recalcitrant species,
embryo banking, a method which also utilizes in vitro culture for recovery germination, is possible. The number of endangered species that will require in vitro methods is estimated to be at least 5,000 worldwide. Further information is needed to identify these species, and the ongoing
collection of information into databases on endangered species and recalcitrant species will help provide this. The costs
of these methods are higher than for traditional propagation and preservation, but they may be necessary for species under
higher threat. The multiplication rate of a culture, as well as the rates of rooting and acclimatization, has a major effect
on the number of transfers needed for producing plants or tissue for banking, and improvements that will increase the efficiency
of these steps can help lower costs. Further research into factors affecting the growth of tissues in vitro, as well as coordination of efforts among institutions with infrastructure for in vitro work, should facilitate the application of in vitro methods to the endangered species that cannot be propagated or preserved using seeds.
KeywordsCost–
In vitro
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Ex situ
–Conservation
In living plant collections, vegetatively propagated accessions are outstanding material with respect to vulnerability and labour amount. This is also true for the main vegetative material, held in the IPK, Gatersleben. A survey of the preservation of potato, garlic and other alliums, mint and yam is given. More than 630 accessions are in slow growth conditions. Amongst them, 99 clones of garlic and 35 of shallot have been tested to be virus-free. Cryopreservation is routinely applied for potato using the droplet method. The cryo-collection contains more than 1000 accessions, a part of which has been integrated from another collection, formerly established at Braunschweig. Cryopreservation of garlic has been used to store the accessions of the European core collection. Cryopreservation is successful in three Dioscorea species using a combination of the original vitrification with the droplet method. Investigations about morphogenesis and ultrastructural cell parameters before, during and after cryopreservation were included in these activities.
One millimeter sized shoot-tips excised from rooted in vitro plants of the genera Musa and Ensete were successfully cryopreserved with the droplet vitrification technique. We show that the loading phase can be prolonged up to 7 h and that the optimal length of PVS2 treatment is 30–50 min at 0 °C. Ultra rapid freezing and thawing rates proved to be essential for high and reproducible post-thaw regeneration rates. This results in an increase of 23–46% compared to the normal cryovial freezing protocol. When the optimal procedure was applied to 56 accessions belonging to eight different genomic groups of Musa spp. and one Ensete spp., an average of 52.9% post-thaw regeneration was obtained. These results were relatively genotype independent. Only wild diploid Musa acuminata accessions proved to be somewhat more recalcitrant towards cryopreservation though an acceptable average regeneration rate of 39% was still obtained.
The ability of seeds to survive desiccation is an important functional trait and is an integral part of plant regeneration ecology. Despite this, the topic has received relatively little attention from ecologists. In this study, we examine the relationships between seed desiccation tolerance and two important aspects of plant regeneration ecology: habitat and dormancy. This is done by comparative analysis of a data set of 886 tree and shrub species from 93 families.
The proportion of species displaying desiccation sensitive seeds declines as the habitat becomes drier, and possibly also cooler, although the latter observation requires cautious interpretation. Desiccation sensitivity is most common in moist, relatively aseasonal vegetation zones, but is infrequent in, though not absent from arid and highly seasonal habitats.
The highest frequency of desiccation sensitivity occurs in non‐pioneer evergreen rain forest trees, although 48% of the species examined have desiccation tolerant seeds. In contrast, all pioneer taxa within the data set have drying tolerant seeds.
Desiccation sensitivity is more frequent in seeds that are non‐dormant on shedding ( c. 31%), than dormant ( c. 9%). Highest frequencies of drying tolerance occur in seeds with physical or combinational dormancy, at 99% and 100%, respectively.
Although there is an association between non‐dormancy and desiccation sensitivity in both tropical and temperate zones, the relationship does not appear to be causal.
Working from the hypothesis that seed desiccation sensitivity represents a derived state in extant species, we use the results to investigate and discuss possible ecological trade‐offs and associated fitness advantages. These may explain the hypothesized repeated loss of this trait. The frequent association between large seed size and desiccation sensitivity is also considered.