The distribution and abundance of Boloria selene (small pearl-bordered fritillary butterfly, SPBF) declined over recent decades in many parts of the UK. Availability of food plants, especially marsh violet (Viola palustris), for the caterpillars of the SPBF has been identified as one of the major causes of this decline. To achieve augmentation of existing colonies and develop new populations of SPBF large numbers of marsh violet propagules were required specifically to feed the larvae. The main objectives of the study were to produce thousands of good quality marsh violet propagules to restore selected habitats in the Heart of Durham, Northern England, using in vitro methods. Preliminary trials showed that in vitro multiplication of seedlings from wild collected seeds through conventional agar-based cultures was lengthy, expensive and turned out to be a non-viable route to achieve the objectives. This study explored the potential of bioreactor-based cloning and cost-effective one step rooting and weaning. Robust propagules, ready for transplantation following rapid propagation and one step rooting and weaning in vitro, were raised in a plug system for transplantation and establishment under field conditions. This was achieved by using simple and cost-effective methods to support the large-scale restoration exercise using 14,000 propagules. Application of high throughput micropropagation and low cost one step weaning systems for time-bound conservation and restoration projects are discussed in detail. This research highlights the important role of in vitro methods to support integrated biodiversity conservation of a native larval host plant and threatened butterfly.

The inselbergs of the Central Highlands of Madagascar are one of many ‘micro-hotspots’ of biodiversity on the island, particularly for Orchidaceae. In this region are several genera that have a large number of endemic species that are in serious decline or edging towards extinction. Studies relating to diversity of orchids and their fungal partners (both mycorrhizal and non-mycorrhizal root associates) deserve more attention, as climate change and human induced decline in resilience of species in the wild is at an all-time high. Identification of mycorrhizal fungi (MF) via conventional seed baited-protocorms has limitations for large scale studies and its application for time-bound conservation projects. The paper describes the value of understanding fungal diversity in the roots of orchids at different stages of maturity. The first part of the study was a preliminary investigation mainly to identify culturable Rhizoctonia endophytes, and the second part looked at all life forms of available taxa together with associated soil characteristics. We isolated and identified 19 putative MF from 18 of the 50 taxa spread over an area of 250 sq. km, covering three life forms, growth phases of the orchid taxa, and habitat types. In the rest of the taxa, we were unable to detect any putative MF, but had varying numbers of non-mycorrhizal endophytes. We also found that diversity of putative MF was higher in plants from soils with the lowest P levels recorded. Putative mycorrhizal OTUs were predominantly from the Tulasnella lineage, followed by Ceratobasidium and Serendipita. Within a small subset of samples, a difference in colonised endophytes depending on the collection season was observed. In vitro germination studies using 10 OTUs of mycorrhizal fungi in 14 orchid species showed mostly generalist associations. When orchid seed and fungal sources were studied irrespective of habitat, life form, and distance from each other (orchid seed and fungal source), compatibility for symbiotic seed germination was observed in most cases. Issues with the identification of compatible MF and symbiotic system of seed germination are discussed.

About 90% of Cynorkis species are endemic to the biodiversity hotspot of Madagascar. This terrestrial habitat-specific genus received little study for fungal diversity to support conservation. We evaluated the diversity of culturable fungi of 11 species and soil characteristics from six sites spanning a >40 km radius in and along the region’s inselbergs. Peloton-forming fungi were grown in vitro from root/protocorm slices and positively identified using DNA sequencing. The fungal diversity was then correlated with soil pH, NO3-N, P, and K. All species harbored either putative mycorrhizal associates in the Rhizoctonia complex or Hypocreales fungi. Tulasnella Operational Taxonomic Units (OTUs) were most prevalent in all soil types while Serendipita OTUs were found in species inhabiting granite/rock outcrops in moist soil (seepage areas). Most Cynorkis species were present in soil with low NO3-N and P levels with diversity of mycorrhizal fungi inversely correlated to NO3-N levels. Of the different orchid life stages sampled, only one species (Cynorkis fastigiata) yielded putative mycorrhizal fungi from juvenile stages. As diversity of mycorrhizal fungi of Cynorkis spp. was negatively correlated with NO3-N, and majority of the studied taxa were found in soils with low NO3-N and P contents, reintroduction studies must include analysis of N and P in detail. For the first time, we showed that the assemblage of culturable fungi in the roots of habitat-specific species of Cynorkis (Orchidaceae) are intimately tied to specific soil characteristics.

Orchid mycorrhizal fungi (OMF) are critical for seed germination and maintaining natural populations of orchids, yet the degree of specificity of most orchids to their mycorrhizal associates remains unknown. Many orchids are at risk of extinction, whether generalists or specialists, but orchid species of narrow fungal specificity are arguably under increased threat due to their requirement for specific fungal symbionts. This study characterises the fungi associated withAerangis ellisii, a lithophytic orchid from a site in the Central Highlands of Madagascar. Culturable OMF isolated from spontaneous protocorms of this species from the wild were used for seed germination. In vitro germination and seedling development ofA. ellisiiwere achieved with fungi derived fromA. ellisiiand an isolate from a differentAerangisspecies 30 km away. The significance of these findings and their importance to conservation strategies for this species and otherAerangisspp. is discussed. These results have important implications for the conservation ofA. ellisiipopulations in Madagascar.

Cephalanthera rubra (L.) Rich., Red Helleborine, is a widespread orchid in Europe but known only from three very small populations in England. These populations are in decline with no natural seed setting for more than a decade. The species may become extinct in the UK soon unless viable strategies are in place for ex situ conservation, especially the use of symbiotic propagation. Because of the fragile nature of the populations in England mycorrhizal fungal diversity study is not feasible. Therefore, to understand the factors needed for healthy Red Helleborine populations, soil characteristics and diversity of culturable root-derived fungi of the populations from a small area in the Loire Valley in France were studied. The main objectives of the study were: (1) Which culturable mycorrhizal fungi associated with C. rubra roots and (2) To what extent is variation in fungal communities related to variation in soil characteristics? Here, we report a significant difference in diversity of culturable mycorrhizal and non-mycorrhizal fungi depending on soil pH and phosphorus content. Mycorrhizal associations were favoured by plants in locations with low soil nutrient availability and comparatively higher pH. Our study shows that mycorrhizal fungi, both ecto and endo, can be cultured from roots of plants at different maturity stages.

Botanical, historical, and archaeological collections have been the source of extraordinarily long-lived seeds, which have been used to revive extinct genotypes or species. The longest-lived example of a viable seed of known age is the date palm, Phoenix dactylifera L., of which an estimated 2000-year-old seed was germinated in 2005. Seed longevity is important for agriculture and biodiversity conservation, and understanding the basis for the extraordinary longevity of seeds from botanical collections could help improve seed banking technology. In this work, we studied the viability and structural features of date palm seeds collected in Baghdad in 1873 and stored in the Economic Botany Collection (EBC) at the Royal Botanic Gardens, Kew, and seeds collected in 2004 and stored dry at -20°C in the Millennium Seed Bank (MSB). Viability was studied by attempted seed germination and in vitro culture of embryos, and structural features were studied by X-rays, transmission electron microscopy, and differential scanning calorimetry. We found that the seeds preserved in the MSB did not decrease in viability, with ultrastructural features similar to those in freshly harvested seeds. In contrast, the 144-year-old seeds were dead, and large ultrastructural changes were observed, particularly in the storage lipids (size, distribution, and melting properties) and other storage constituents. These results contrast with previous reports that date seeds could remain viable for ∼2000 years in uncontrolled storage environments. We did not find that the postharvest treatment of the EBC seeds in the 19th century, or their storage conditions at Kew, was more deleterious than that which was likely encountered by the ∼2000-year-old seeds. These results highlight the role of well-documented collections in establishing whether reports of extraordinary longevity are ordinarily repeatable.

Tropical orchids need more study with respect to their mycorrhizal associations. For researchers in distant countries who aspire to study these orchids augmenting their conservation, the great distances involved, coupled with limited funds, pose formidable challenges. These challenges are sometimes exacerbated by political unrest, delays in securing permits, unexpected hardships, and the risk that the biological samples collected (e.g., roots harboring mycorrhizal fungi) will not survive long-distance transport.

Background: Lack of phenological information and efficient collecting methods are considered impediments for orchid seed collecting. This leads to opportunistic collecting as part of general seed collecting schedules that may last few weeks especially in remote areas. The study explored the feasibility of collecting near mature and immature seeds to support conservation action plans. Mature, near mature and immature seeds of orchids were collected from the wild in the Central Highlands of Madagascar (CHM). Seed capsules were collected in sterile culture medium in the wild, to prevent deterioration of seeds inside the capsule after collecting, later to be cultured under laboratory conditions.Results: Seed capsules collected by the in vitro collecting (IVC) method were kept in very good condition for up to 4 weeks before germination under in vitro conditions. Significantly faster and higher germination rate (p < 0.001) than mature seeds was observed in a number of taxa collected during a 3 year-long study. In some taxa even immature seeds, with no apparent sign of testa covering the embryo, germinated following IVC where mature seeds failed to germinate.Conclusions: We propose that IVC method has potential to complement conventional seed collecting by increasing the germplasm that can be used in integrated conservation action plans. Improvements can be made in developing collections for taxa from biodiversity hotspots and remote areas where collecting requires considerable resources. This method can further be used on a wider selection of plants from different geographic areas and on embryo rescue programmes for economically important plants.

The Orchid flora of Madagascar is one of the most diverse with nearly 1000 orchid taxa, of which about 90 % are endemic to this biodiversity hotspot. The Itremo Massif in the Central Highlands of Madagascar with a Highland Subtropical climate range encompasses montane grassland, igneous and metamorphic rock outcrops, and gallery and tapia forests. Our study focused on identifying culturable mycorrhizae from epiphytic, lithophytic, and terrestrial orchid taxa to understand their diversity and density in a spatial matrix that is within the protected areas. We have collected both juvenile and mature roots from 41 orchid taxa for isolating their orchid mycorrhizal fungi (OMF), and to culture, identify, and store in liquid nitrogen for future studies. Twelve operational taxonomic units (OTUs), of three known orchid mycorrhizal genera, were recognized by analysis of internal transcribed spacer (ITS) sequences of 85 isolates, and, by comparing with GenBank database entries, each OTU was shown to have closely related fungi that were also found as orchid associates. Orchid and fungal diversity were greater in gallery forests and open grasslands, which is very significant for future studies and orchid conservation. As far as we know, this is the first ever report of detailed identification of mycorrhizal fungi from Madagascar. This study will help start to develop a programme for identifying fungal symbionts from this unique biodiversity hotspot, which is undergoing rapid ecosystem damage and species loss. The diversity of culturable fungal associates, their density, and distribution within the Itremo orchid hotspot areas will be discussed.

In 1996 the IUCN/SSC Orchid Specialist Group published a Status Survey and Conservation Action Plan that included a number of recommendations designed to address the problem of a projected imminent and rapid decline of species. Orchids remain subject to a multiplicity of threats in their natural habitats and, in addition to the usual suspects of habitat loss and land conversion, climate change has exerted a measurable effect on some orchid populations. Collection of orchids for traditional medicine is having a significant effect both within China and surrounding countries. Some progress has been made in achieving the targets set for Red Listing of endangered species in some countries, but other countries are lagging behind. In addition to successful initiatives in preserving natural habitats, a number of projects around the world have demonstrated that it is possible both to bring endangered species into cultivation and successfully reintroduce them into the wild. Orchid Seed Stores for Sustainable Use, with its goal of storing seeds representing a minimum of 1000 orchid species in institutes in at least 30 countries over the next five years, provides an example of what can be achieved by a small, dedicated group determined to share their technical expertise and develop a deeper understanding of the underlying science. There is, however, also an urgent need to promote awareness in the wider community about the importance of all sorts of biodiversity (not only orchids) through educational programs and involve people from as wide a range of backgrounds as possible.