Bryophyte spores are known for long-distance dispersal and tolerance to adverse conditions, but their comparative longevity remains poorly understood. In this work we assessed whether moss spores are as long-lived as spores and seeds of other land plants. Spores of Funaria hygrometrica, Lewinskya acuminata, L. iberica and Ulota crispula were subjected to accelerated aging (AA: 45 °C, 60% relative humidity [RH]) and dry storage (15% RH, 20 °C) with periodic germination tests. Different maturity stages of F. hygrometrica were also included. Estimated initial viability constant (Ki), standard deviation of spore deaths’ distribution in time (σ) and time for viability to drop to 50% (P50) were determined by probit analysis from spore survival curves. Overall results showed short spore longevity for all species tested, regardless of storage conditions. P50 ranged from 0.4 to 5.7 days (AA) and 58–150 days (dry storage), indicating faster deterioration than seeds or fern spores at the same storage conditions. In the case of F. hygrometrica, spores collected at peak maturity showed the longest longevity under both storage conditions. This study suggest that moss spores may be relatively short-lived, which could be related to their chlorophyllous nature. Ecological and conservation implications of this spore character are discussed.

An estimated 45% of plants are threatened with extinction, with those classified as "Critically Endangered" (CR) by the International Union for Conservation of Nature at the highest risk and in urgent need of ex situ conservation. We performed a gap analysis of CR plants stored in in the Millennium Seed Bank Partnership (MSBP), a global network spanning 101 countries and territories, to assess their current conservation status and guide future seed collection priorities. The MSBP currently holds 2,507 accessions of 474 CR taxa, representing 0.9% of the 52,874 taxa banked and 7.21% of all CR taxa. While most collections have relatively good general information available (information index = 0.81), key aspects such as viability (viability index = 0.31) and genetic diversity (genetic diversity index = 0.31) remain poorly documented. Only 9.49% of conserved CR taxa have sufficient seed numbers (>= 1,050 seeds from >= 50 individuals) for restoration research, and just 5.49% have been collected from >= 5 populations and >= 50 individuals across their native distribution. Overall, 6,120 CR plant taxa remain unbanked, of which 11.58 to 17.17% (depending on model) require alternative conservation methods due to desiccation-intolerant seeds. However, the majority (23.32-68.74% taxa) are likely suitable for traditional seed banking techniques (drying to 15% relative humidity and freezing at -20 degrees C). The desiccation sensitivity of 14.02-65.16% taxa remains unknown, requiring urgent research. Ensuring comprehensive ex situ conservation of CR taxa will require strategic investment and capacity building to meet global biodiversity conservation needs.

BACKGROUND AND AIMS:A quarter of the assessed carnivorous plants (CPs) are threatened with extinction, and the effectiveness of ex situ conservation initiatives must be evaluated to complement in situ conservation efforts. Conventional seed storage (e.g. 15 % relative humidity, -20 °C) is the most common and efficient strategy for plant ex situ conservation, but seeds of diverse CPs might be short lived in these conditions. Thus, there is a need to assess their longevity empirically and comparatively and to evaluate the success of alternative storage options. METHODS:Diverse seed collections were used to assess longevity of seeds of 13 CP species stored (some for 30 years) in various storage conditions: ambient/uncontrolled, conventional and cryogenic. KEY RESULTS:The relative short longevity of CP seeds stored in ambient/uncontrolled and conventional conditions was confirmed for some taxa (e.g. Drosera rotundifolia L.). Nevertheless, despite this potential short longevity, seeds of 64 % of accessions tested retained their initial viability for up to two decades when stored in conventional seed bank conditions. Only some accessions stored for longer times (>25 years) showed significant signs of deterioration, with D. rotundifolia and Drosera anglica Huds. showing a time for viability to fall to 50 % (P50) in the region of other taxa considered medium/short or short lived in comparable cold/dry storage conditions. As an alternative (or complement) to conventional seed banking, cryogenic storage of dry seeds was able to preserve a high germination percentage of seeds of CPs stored for up to two decades. Furthermore, seedlings obtained developed normally into healthy plants when monitored for ≤1.5 years after germination. CONCLUSIONS:Despite the potential short lifespan of seeds of some CP taxa, this paper highlights and supports their routine dry storage in ex situ conservation programmes to support in situ conservation initiatives.

In the 19th century it was proposed that ecophysiology was best studied in regions with extreme climatic conditions. In the present perspective, we argue that perhaps this is more timely than ever. The main reason is the need to improve crops to be simultaneously more productive-due to the increased population-and more stress tolerant-due to climate change. Climate change induces plants to face not just harsh but also 'unexpected' (unpredictable) climatic conditions. In this sense, we hypothesize that 'sherplants', namely plants living in the extremes of plant life (e.g. hot deserts, Arctic and Antarctica, or high elevations) can provide cues on how to break the trade-off between productivity and stress tolerance, as they need to be produced quickly due to the very short growing period while being stress tolerant due to the harsh and unpredictable climate endured during most of the year. We present glimpses of results from three consecutive projects developed over the last 10 years, in which hundreds of species from different regions of the world have been studied. In particular, we propose a pathway for developing 'shercrops' learning from 'sherplants', debate whether some of the already studied species may have really broken the aforementioned trade-off, and present a number of interesting unforeseen discoveries made when studying plants from extreme climates.

This Editorial introduces and contextualizes the papers of the Virtual Special Issue: Plant cryobiotechnology: multidisciplinary and modern research to understand the fundamental basis that allows successful cryopreservation and recovery.

Progressive loss of plant diversity requires the protection of wild and agri-/horticultural species. For species whose seeds are extremely short-lived, or rarely or never produce seeds, or whose genetic makeup must be preserved, cryopreservation offers the only possibility for long-term conservation. At temperatures below freezing, most vegetative plant tissues suffer severe damage from ice crystal formation and require protection. In this review, we describe how increasing the concentration of cellular solutes by air drying or adding cryoprotectants, together with rapid cooling, results in a vitrified, highly viscous state in which cells can remain viable and be stored. On this basis, a range of dormant bud–freezing, slow-cooling, and (droplet-)vitrification protocols have been developed, but few are used to cryobank important agricultural/horticultural/timber and threatened species. To improve cryopreservation efficiency, the effects of cryoprotectants and molecular processes need to be understood and the costs for cryobanking reduced. However, overall, the long-term costs of cryopreservation are low, while the benefits are huge.

Abstract. Ice nucleating molecules (INMs) produced by plant pollen can nucleate ice at warm temperatures and may play an important role in weather and climate relevant cloud glaciation. INMs have also proved useful for mammalian cell and tissue model cryopreservation. The high ice nucleation (IN) activity of some INMs indicates an underlying biological function, either freezing tolerance or bioprecipitation mediated dispersal. Here, using the largest study of pollen ice nucleation to date, we show that phylogenetic proximity, spermatophyte subdivision, primary growth biome, pollination season, primary pollination method, desiccation tolerance and native growth elevation do not account for the IN activity of INMs released from different plant species’ pollen. The results suggest that a polysaccharide present in pollen is produced by plants for a purpose unrelated to ice nucleation has an incidental ability to nucleate ice. This ability may have been adapted by some species for specific biological purposes, producing exceptional ice nucleators. Pollen INMs may be more active, widespread in nature, and diverse than previously thought.

Ice-nucleating macromolecules (INMs) produced by plant pollen can nucleate ice at warm temperatures and may play an important role in weather- and climate-relevant cloud glaciation. INMs have also proved useful for mammalian cell and tissue model cryopreservation. The high ice nucleation (IN) activity of some INMs indicates an underlying biological function, either freezing tolerance or bioprecipitation-mediated dispersal. Here, using the largest study of pollen ice nucleation to date, we show that phylogenetic proximity, spermatophyte subdivision, primary growth biome, pollination season, primary pollination method, desiccation tolerance and native growth elevation do not account for the IN activity of INMs released from different plant species' pollen. The results suggest that these macromolecules are produced by plants for a purpose unrelated to ice nucleation and have an incidental ability to nucleate ice. This ability may have been adapted by some species for specific biological purposes, producing exceptional ice nucleators. Pollen INMs may be more active, widespread in nature, and diverse than previously thought.

Cryopreservation, or the storage at liquid nitrogen temperatures (-196°C), of embryogenic cells or somatic embryos allows their long-term conservation without loss of their embryogenic capacity. During the last decade, protocols for cryopreservation of embryogenic material of woody species have been increasing in number and importance. However, despite the large experimental evidence proved in thousands of embryogenic lines, the application for the large-scale conservation of embryogenic material in cryobanks is still limited. Cryopreservation facilitates the management of embryogenic lines, reducing costs and time spent on their maintenance, thus limiting the risk of the appearance of somaclonal variation or contamination. Somatic embryogenesis in combination with cryopreservation is especially useful to preserve the juvenility of lines while the corresponding clones are being field-tested. Hence, when tree performance has been evaluated, selected varieties can be propagated from the cryostock. The traditional method of slow cooling or techniques based on vitrification are mostly applied procedures. For example, slow cooling methods are widely applied to conserve embryogenic lines of conifers. Desiccation based procedures, although simpler, have been applied in a smaller number of species. Genetic stability of the cryopreserved material is supported by multiloci PCR-derived markers in most of the assayed species, whereas DNA methylation status assays showed that cryopreservation might induce some changes that were also observed after prolonged subculture of the embryogenic lines. This article reviews the cryopreservation of embryogenic cultures in conifers, fruit species, deciduous forest species and palms, including a description of the different cryopreservation procedures and the analysis of their genetic stability after storage in liquid nitrogen.

Recalcitrant seeds are characterized by desiccation and freezing sensitivity, and short storage longevity. These physiological attributes obviate their ex situ conservation in conventional seed banks, where seeds are stored dry at sub-zero temperatures (typically, 15% relative humidity and –20°C) for extended periods of time. Propagation of plants for field collections (e.g., botanical gardens, nurseries, and arboretums) is a valuable ex situ conservation option. However, these collections are relatively costly, require high maintenance, preserve limited genetic diversity and/or are directly exposed to biotic (e.g., pests) and abiotic (e.g., climatic) threats. Therefore, recalcitrant-seeded (RS) species are dependent on cryopreservation for their safe and long-term ex situ conservation. Different explant sources such as whole seeds, zygotic embryos, dormant buds, shoot tips, and pollen, can be used for plant propagation of RS species in field collections as well as for their cryopreservation. The success of the propagation or the cryopreservation of these explants often depends on their developmental status, vigor, and/or tolerance to desiccation and chilling/freezing. These attributes are modulated by the environment where the donor plant grows and we hypothesize that climate change, by affecting these biological attributes, would impact the success of explant propagation and cryopreservation. To support this hypothesis, we have reviewed how temperature changes and drought, the two main climate change scenarios, affect the main biological attributes that are directly involved in the success of ex situ conservation of tropical and temperate RS species. In general, increases in temperature and drought will negatively affect plant development in field collections and the quality of the explants used in cryopreservation. Consequently, field collections of RS species may need to be moved to more suitable places (e.g., higher latitudes/altitudes). Additionally, we may find a reduction in the success of cryopreservation of RS species germplasm directly harvested from field collections. However, we cannot always generalize these effects for all species since they often depend on the origin of the species (e.g., tropical and temperate species tend to respond to climate change differently), the genotype, the adaptive genetic potential of each population, and the severity of the environmental change. On the other hand, the increase in temperatures and water stress in donor plants at high-latitude areas and also some tropical environments may favor the production of seeds and seedlings better adapted to drying, and hence, increase the success of plant propagation and zygotic embryo cryopreservation.

Ageing in dry chlorophyllous propagules is leaded by photooxidation through the photosynthetic machinery, but why species differ in longevity and the ageing mechanisms of when light and oxygen are absent are unknown. We hypothesize that the cellular antioxidant capacity is key for the inter- and intra-specific differences in the ageing process. We have tested this hypothesis in chlorophyllous spores of two ferns. They were subjected to four different storage regimes resulting from light/dark and normoxia/hypoxia combinations. Lipophilic and hydrophilic antioxidants, reactive oxygen species (ROS), and photosynthetic pigments were analysed in parallel to germination and the recovery of Fv/Fm over a storage period of up to 22-months. We show that light and oxygen accelerate the ageing process, but their mechanisms (ROS, increase, antioxidant capacity decrease, loss of efficiency of the photosystem II, pigment degradation) appear the same under all conditions tested. The end of the asymptomatic phase of longevity, when a sudden drop of germination occurs, seems to be determined by a threshold in the depletion of antioxidants. Our results support the hypothesis that ageing kinetics in dry plant propagules is determined by the antioxidant system, but also suggests an active role of the photosynthetic machinery during ageing, even in darkness and hypoxia.

The lifespan or longevity of a seed is the time period over which it can remain viable. Seed longevity is a complex trait and varies greatly between species and even seed lots of the same species. Our scientific understanding of seed longevity has advanced from anecdotal 'Thumb Rules,' to empirically based models, biophysical explanations for why those models sometimes work or fail, and to the profound realisation that seeds are the model of the underexplored realm of biology when water is so limited that the cytoplasm solidifies. The environmental variables of moisture and temperature are essential factors that define survival or death, as well as the timescale to measure lifespan. There is an increasing understanding of how these factors induce cytoplasmic solidification and affect glassy properties. Cytoplasmic solidification slows down, but does not stop, the chemical reactions involved in ageing. Continued degradation of proteins, lipids and nucleic acids damage cell constituents and reduce the seed's metabolic capacity, eventually impairing the ability to germinate. This review captures the evolution of knowledge on seed longevity over the past five decades in relation to seed ageing mechanisms, technology development, including tools to predict seed storage behaviour and non-invasive techniques for seed longevity assessment. It is concluded that seed storage biology is a complex science covering seed physiology, biophysics, biochemistry and multi-omic technologies, and simultaneous knowledge advancement in these areas is necessary to improve seed storage efficacy for crops and wild species biodiversity conservation.

Many useful plant species with potential for plant-based bioregenerative life support systems produce extremophile seeds with tolerance to multiple stressors, including desiccation, which allows for their transport through space in a dried state. However, other valuable species produce desiccation-sensitive seeds or are propagated clonally, and life sciences research in space has not yet addressed the challenge of alternative transport methods in microgravity for such material. Although liquid nitrogen storage is used on Earth for desiccation-sensitive germplasm, it poses atmospheric leakage problems to crewed spacecraft and therefore liquid nitrogen-free cryogenic freezing could be an alternative. Another promising approach is slow growth tissue culture, with subculture intervals extended to months or years through the precise control of the culture environment. Whilst the design of innovative systems for the transport of species with desiccation-sensitive germplasm will be demanding, the prospect still remains for their successful growth beyond Earth.

Haematoccocus lacustris (previously H. pluvialis) is a desiccation-tolerant unicellular freshwater green alga. During acclimation to desiccation, astaxanthin-rich lipid bodies and low-molecular-weight antioxidants (α-tocopherol, glutathione) accumulate, while the chloroplast area and chlorophyll contents decrease, which may facilitate desiccation tolerance by preventing damage mediated by reactive oxygen species (ROS). Here, we investigated the influence of moisture, light, oxygen, and temperature on redox homeostasis and cell longevity. Respiration and unbound freezable water were detectable in cells equilibrated to ≥90% relative humidity (RH), a threshold above which viability considerably shortened. At 92.5% RH and 21 °C, antioxidants depleted over days as cells lost viability, especially in an oxygen-rich atmosphere, supporting the role of ROS production in uncoupled respiration in viability loss. At 80% RH and 21 °C, redox homeostasis was maintained over weeks, and longevity was less influenced by oxygen. Light and oxygen was a lethal combination at 92.5% RH, under which pigments bleached, while in the dark only astaxanthin bleached. Viability positively correlated with glutathione concentrations across all treatments, while correlation with α-tocopherol was weaker, indicating limited viability loss from lipid peroxidation at 80% RH. In cells equilibrated to 50% RH, longevity and redox homeostasis showed strong temperature dependency, and viability was maintained at sub-zero temperatures for up to three years, revealing cryogenic storage to be an optimal strategy to store H. lacustris germplasm.