Plant cryobanks play a significant role in modern science and breeding. They contribute to the recovery of lost species, the emergence of new plant varieties, and help preserve and explore the diversity of the plant world. The IPPRAS Cryobank collection is constantly supplemented with new samples, while, at the same time, the stored samples are being monitored. In order to test seed germination, seeds of Allium and Veratrum species were thawed. Rare Allium species seeds, such as A. nutans, A. schoenoprasum, and A. victorialis were stored in liquid nitrogen for 17, 19, and 30 years, respectively. Long-term cryopreservation decreased germination rates for A. nutans from 96.55 to 50.00%, for A. schoenoprasum from 72.00 to 62.75%, and for A. victorialis from 90.00 to 83.05%. Seeds of a rare medicinal species, Veratrum lobelianum, were stored in liquid nitrogen for 18 years; the seed germination rate during this storage period has been significantly decreased from 75.00 to 14.81%. V. nigrum seeds were also collected and frozen in liquid nitrogen for 3 days. Short-term cryopreservation did not result in a statistically significant change in germination rates (from 79.71 to 82.69%). The seeds of an endangered ornamental species, Cypripedium calceolus, were collected and kept frozen for 3 days. After cryopreservation, the seeds were planted on three different media, as follows: ½ MS, MS with 10% coconut milk, and BM1. On ½ MS medium, 24.98% seeds formed protocorms, while on MS medium with 10% coconut milk, this number was 10.02%, and on BM1 medium, it was 15.02%, respectively; however, after 2.5 months, all of the protocorms died. Thus, it appears that the existing protocol for seed cryopreservation of C. calceolus needs further improvement. The size, weight, and free water content (WC) of six previously cryopreserved Stipa species and three Allium species were measured. For all the Allium and Stipa species studied, we found no correlation between seed size, WC, and cryotolerance. We also found no correlation between the life form, which reflects the water requirement of the species, and cryotolerance.

Estimating effective population size (N e) is important for theoretical and practical applications in evolutionary biology and conservation. Nevertheless, estimates of N e in organisms with complex life-history traits remain scarce because of the challenges associated with estimation methods. Partially clonal plants capable of both vegetative (clonal) growth and sexual reproduction are a common group of organisms for which the discrepancy between the apparent number of individuals (ramets) and the number of genetic individuals (genets) can be striking, and it is unclear how this discrepancy relates to N e. In this study, we analysed two populations of the orchid Cypripedium calceolus to understand how the rate of clonal versus sexual reproduction affected N e. We genotyped >1000 ramets at microsatellite and SNP loci, and estimated contemporary N e with the linkage disequilibrium method, starting from the theoretical expectation that variance in reproductive success among individuals caused by clonal reproduction and by constraints on sexual reproduction would lower N e. We considered factors potentially affecting our estimates, including different marker types and sampling strategies, and the influence of pseudoreplication in genomic data sets on N e confidence intervals. The magnitude of N e/N ramets and N e/N genets ratios we provide may be used as reference points for other species with similar life-history traits. Our findings demonstrate that N e in partially clonal plants cannot be predicted based on the number of genets generated by sexual reproduction, because demographic changes over time can strongly influence N e. This is especially relevant in species of conservation concern in which population declines may not be detected by only ascertaining the number of genets.

In this article we present and discuss the main factors that threaten natural populations of Cypripedium calceolus (lady\'s slipper orchid) in Europe, and we propose conservation strategies and directions for protective actions of its population on a regional scale. European C. calceolus populations have decreased significantly in the last two decades, in both number and size. A key result of the present study is an evaluation of the effectiveness of the Natura 2000 network across the European Union (EU) countries. Northern and/or mountainous countries present higher percentages of potentially suitable areas within the Natura 2000 network. Finland and the United Kingdom are the exceptions to this rule. It is predicted that, due to global warming, the coverage of niches suitable for C. calceolus will decrease in countries in which now-healthy colonies exist. However, as plant species can occur in micro-sites with suitable environmental conditions (e.g., microclimate, vegetation, soil factors) which cannot be predicted as suitable at coarser spatial resolutions, conservation efforts should be focused on management of local healthy populations. For the effective protection of C. calceolus in Natura 2000 sites, the participation of experts in botany, including orchid biology, is necessary at several stages.

Floral scent, a key mediator in plant-pollinator interactions, varies not only among plant species, but also within species. In deceptive plants, it is assumed that variation in floral scents and other traits involved in pollinator attraction is maintained by negative frequency-dependent selection, i.e., rare phenotypes are more attractive to pollinators and hence, have a higher fitness than common phenotypes. So far, it is unknown whether the rarity of multivariate and/or continuous floral scent traits influences the pollination success of flowers. Here, we tested in the deceptive orchid Cypripedium calceolus, whether flowers with rarer scent bouquets within a population have a higher chance to getting pollinated than flowers with more common scents. We collected the scent of more than 100 flowers in two populations by dynamic headspace and analyzed the samples by gas chromatography coupled to mass spectrometry (GC/MS). From the same flowers we also recorded whether they set a fruit or not. We introduced rarity measures of uni- and multivariate floral scent traits for single flowers, which allowed us to finally test for frequency-dependent pollination, a prerequisite for negative frequency-dependent selection. Our results do not show rarity has an effect on the likelihood to set fruits in neither of the two populations and in none of the scent characteristics analyzed. Hence, there is no evidence of negative frequency-dependent pollination mediated by the floral scent of C. calceolus. We discuss that our approach to determine rarity of a scent is applicable to any univariate or multivariate (semi)quantitative trait.

The complete chloroplast genome of Cypripedium calceolus, a rare species in the family Orchidaceae was reported in this study. The genome size is 175,122 bp in length, and contains four sub-regions: 97,486 bp of large single copy (LSC) and 22,260 bp of small copy (SSC) regions, separated by 27,688 bp of inverted repeat (IR) regions. A total of 133 genes were annotated, including 87 protein-coding genes, 38 tRNA genes and 8 rRNA genes. The GC content of this cp genome is 34.36%. Phylogenetic analysis revealed a close relationship between C. calceolus with C. japonicum and C. formosanum. This is the first complete cp genome for C. calceolus that would be useful for conservation and phylogenetic studies of this species.