Fern spores and pollen are haploid plant germplasm of microscopic nature that can be used to regenerate full plants through germination (fern spores) or to fertilize seed-bearing plants through breeding programs (pollen). Due to their short life span in conventional storage (i.e., dry at -20 degrees C), the use of cryopreservation has been indicated for long-term ex situ conservation. While fern spores of most species and pollen from many seeded plants tolerate desiccation and can be stored dry at liquid nitrogen temperatures, some pollen is desiccation sensitive, and cryopreservation protocols require controlled drying and cooling and some level of cryoprotection. In this chapter we describe the cryopreservation process for fern spores used in the Millennium Seed Bank of Royal Botanic Gardens, Kew, including some details of the fern spores harvest and cleaning methods. In addition, two protocols for pollen cryopreservation are described, one generic for desiccation-tolerant pollen that can be used for multiple species and one specific for a desiccation sensitive pollen (Zea mays).

Know the breeding system of endemic plants is important to design conservation strategies. Translocations are actions to improve the survival prospects of the species, but nowadays there are only a few studies that analyse their success and make a comparison between translocation and the natural populations. Dianthus morisianus is a threatened narrow endemic plant species growing on sand dunes in SW Sardinia (Italy). The objective of this study was to assess the breeding system, the presence of inbreeding depression and pollen limitation, as well as the success of the plant translocation. All these results were compared with those from the single natural population. The breeding system was tested through five pollination treatments and the reproductive success was analysed by the fruit set, seed set, seed weight, germination and mortality rate. The translocated population behaved like the natural one on fruit and seed formation. Autonomous self-pollination was lower than the other treatments regarding fruit set and seed/ovule ratio in the two studied populations. The species is self-compatible and presents partial self-fertility. The selfing rate was higher in the translocated population and the inbreeding depression presented low values for the natural population, while the translocated population presented negative values. Neither of the populations suffered pollen limitation. The species did not present reproductive problems and it is pollinator dependent. Moreover, the translocated population demonstrated high success after five years, as an increase of the population area and new recruited plants was observed; the offspring were able to flower, fruiting and reproduce. This translocation success increases the survival prospects of the species.

Oaks (genus Quercus) are dominant and iconic trees in most European, American, Asian and North African forests, from cool temperate to tropical environments. There are at least 600 species of Quercus globally with high ecological and economical importance. However, 45% of the species evaluated by IUCN are considered threatened, being affected by habitat destruction or diseases and pests, such as acute oak decline or chronic oak dieback in the UK. As a result, conservation of oaks is becoming increasingly important for many countries. Oak seeds are desiccation sensitive (i.e., recalcitrant), and conventional seed bank strategies are not suitable for their long-term preservation. Tissue culture is a useful tool for Quercus species conservation, supporting micropropagation and in vitro conservation. However, cryopreservation is accepted as the most favourable and cost-effective option for oak long-term conservation ex situ. Numerous tissues can be used as germplasm to preserve the genetic diversity of oaks, including winter dormant buds, shoot tips, plumules, embryogenic lines, somatic embryos and embryonic axes. Pollen cryopreservation could also be used as a complementary technology to support conservation and breeding programmes. Interestingly, whilst the seeds of oak are recalcitrant it appears that the pollen has much greater desiccation tolerance. Seed embryonic axes (with shoot and root meristems) are the preferred explants for oak preservation as high genetic diversity can be captured and axes can be grown into full plants with relatively simple micropropagation procedures. However, successful cryopreservation depends on controlling many factors, including physical damage to the embryo during excision, oxidative stress associated to excision and cryopreservation procedures, differential response of shoot and root meristems (within and amongst species) to the stresses imposed by partial desiccation and cooling, and the need to improve in vitro growth and acclimation procedures. Here we review the main cryobiotechnological options for the successful banking of species in the genus Quercus and the needs for future research are highlighted.

The reproductive success of a plant species depends to some extent on its flowering phenology. Thus, it is important to understand the flowering period, the flowering intensity and the synchrony of flowering to determine their respective influence on the reproductive success of the plant species. This study was conducted on the only natural population of Dianthus morisianus and on the first attempted translocation of the species. The aim of this study was to analyse the flowering phenology of the two populations to understand each and compare them. To analyse the flowering phenology, the initiation date, the intensity, the maximum flowering moment, the synchrony and the duration of flowering were tested. The studied plants had different flowering durations between years and the intensity and number of stems were significantly lower in the offspring of translocated plants than the natural plants. The early flowering plants had a longer flowering period and produced more flowers than those that flowered later. The offspring of the translocated population were able to flower, fruit and reproduce. They presented high flowering synchrony and flowered at the same time as the natural plants. This can increase the attractiveness for pollinators and facilitate exchange pollen with the natural plants. After five years, the success of the translocation can, thus, be asserted. This new path increases the genetic diversity of the species and its distribution.

Dianthus morisianus Vals. (Caryophyllaceae) is a narrow endemic species of Sardinia, growing with only one natural population on the Portixeddu coastal dune system (southwest Sardinia). In this work we investigated the ex situ phenology and the breeding system through hand-pollination experiments to investigate whether this endemic plant presents inbreeding depression. D. morisianus had a floral lifespan of a week; the maximum flowering moment occurred in June and the cultivated plants presented high flowering synchrony. Among several traits measured, there were only differences in fruit set and first leaf appearance between geitonogamous and xenogamous treatments, and we detected differences in the number of seeds and first leaf appearance in accordance to the origin of the mother plant (mother effect). The plants cultivated ex situ present inbreeding depression only in germination time (T-50), and the cumulative inbreeding depression was negligible. D. morisianus is self-compatible and presents facultative xenogamy as its breeding system. Our data suggest that inbreeding depression should not be a major issue in D. morisianus. The results of this study are of value for further conservation actions on natural population of this threatened endemic plant. (C) 2015 Elsevier GmbH. All rights reserved.

The Aichi Biodiversity Target 12 aims to prevent the extinction risk of known threatened species and to improve their conservation status by 2020. We present the integrated strategy implemented in the last 10 years for the keystone plant species of Sardinia (Italy, W. Mediterranean Basin), which includes the following activities: conservation status assessment (following the IUCN protocol), ex situ conservation, in situ monitoring and active protection measures. To date, an average of 51.8% of keystone plant species have been subjected to the latter first three activities while, due to the higher costs, only few active conservation measures have been carried out. Considering the activities realised since 2004, we have also predicted the conservation effectiveness towards 2020 and have elaborated an index to evaluate it. Halfway through the strategic plan, we argue that more efforts are needed to guarantee the effective conservation of all threatened plants in Sardinia.