Comparative Biology of Cycad Pollen, Seed and Tissue - A Plant Conservation Perspective.

Cycads are the most endangered of plant groups based on IUCN Red List assessments; all are in Appendix I or II of CITES, about 40% are within biodiversity 'hotspots,' and the call for action to improve their protection is long-standing. We contend that progress in this direction will not be made until there is better understanding of cycad pollen, seed and tissue biology, which at the moment is limited to relatively few (<10%) species. We review what is known about germplasm (seed and pollen) storage and germination, together with recent developments in the application of contemporary technologies to tissues, such as isotype labelling, biomolecular markers and tissue culture. Whilst progress is being made, we conclude that an acceleration of comparative studies is needed to facilitate the integration of in situ and ex situ conservation programmes to better safeguard endangered cycads.

Ageing kinetics of fern chlorophyllous spores during dry storage is determined by its antioxidant potential and likely induced by photosynthetic machinery.

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.

Comparison of seed and seedling functional traits in native Helianthus species and the crop H. annuus (sunflower).

Seed functional traits of native Helianthus species contribute towards ecosystem services but limitations to their use in managed programmes exist. Many perennial Helianthus possess seed dormancy. The ability for germination to occur under different temperature and drought conditions, as well as the capacity of germinated seeds to convert into normal seedlings is rarely considered. Our aim was to identify and quantify these constraints through functional trait analyses. In five seed lots of native Helianthus (four perennial and one annual) and five genotypes of sunflower (H. annuus) for comparison, dormancy, thermal and hydro thresholds and times, morphology, mass, oil content and conversion into normal seedlings were quantified. The influence of the seed collection site environment on these traits was also explored. Seed dormancy of the perennial species was overcome by scarification followed by germination in 5 mm GA3 . Thermal and hydro-time analyses revealed slower germination for the native seed lots (>1350 °Ch) in comparison to the sunflower genotypes (<829.9 °Ch). However, native seed lots had a higher capacity to convert into normal seedlings at high temperatures and low water potentials than sunflower genotypes. For the native seed lots, the average monthly temperature of the collection site was negatively correlated with thermal time. Variability in seed functional traits of native Helianthus and greater capacity for germinated seeds to convert into normal seedlings suggests they are better equipped to cope with high temperature and drought scenarios than sunflower. Effective dormancy alleviation is required to facilitate the use of native Helianthus species.

Integration of genetic and seed fitness data to the conservation of isolated subpopulations of the Mediterranean plant Malcolmia littorea.

Plant autoecology and population genetics provide a perspective on the likelihood of natural regeneration, which is critical when designing conservation strategies for endangered species. The threatened coastal plant Malcolmia littorea (Brassicaceae) was sampled across its European distribution and studied for genetic diversity and seed fitness, with the aim of providing information for the conservation of isolated and declining populations. Nine microsatellite markers (five chloroplast and four nuclear) were analysed to assess population genetic diversity and structure and to conduct a spatial analysis using the software DIVA-GIS. Germination percentages and rates were assessed by incubating the seeds under eight constant temperatures (0-27 °C). The genetic diversity was found to be similar among subpopulations (chloroplast H = 0.04-0.17; nuclear Ho  = 0.20-0.37), with no correlation between subpopulation diversity and the area of occupancy (AOO). The subpopulations were found to be clustered in three genetic groups, and three of them were identified as conservation priorities due to their unique genetic composition. The germination study revealed a significant influence of the maternal environment and AOO on seed germination, with the smaller subpopulations showing lower germination percentages (P < 0.05). These results highlight the importance of obtaining information on isolated subpopulations through different experimental approaches (e.g. seed germination plus population genetics) to enable planning of effective conservation actions. For M. littorea, seed collection for both in situ and ex situ conservation should take into account the local adaptation of the subpopulation and the genetic structure of the species.

Alternating temperature combined with darkness resets base temperature for germination (Tb ) in photoblastic seeds of Lippia and Aloysia (Verbenaceae).

Thermal time models for seed germination assume a continuum of rate responses in the sub-optimal temperature range. Generally, the models describe germination performance in non-dormant seeds at constant temperatures, yet alternating temperature (AT) is a feature of many natural environments. We studied the possible interacting effects of AT on germination progress in photoblastic seeds of three aromatic-medicinal Verbenaceae species in the genera Lippia and Aloysia. For Lippia turbinata f. turbinata and L. turbinata f. magnifolia seed, germination only occurred in light conditions, while for L. integrifolia and Aloysia citriodora it was significantly higher in the light than in darkness. Although relative light germination (RLG) was not different between constant and AT in the sub-optimal range, AT raised the base temperature for germination progress (Tb ) from ca. 3-6 °C in constant temperature to 7-12 °C in AT. Among the species, thermal time for 50% seed germination [θT(50) ] was 55-100 °Cd at constant temperature. Although AT resulted in slight modifications to θT(50) , the germination rate at comparable average temperatures in the sub-optimal range was slower than under constant temperatures. For all species, the proportion of germinated seeds was similar for constant and AT. Our results suggest that an interaction between cool temperature and darkness during AT treatment limits the temperature range permissive for germination in these positively photoblastic seed, reflecting both close adaptation to the natural ecology and niche requirements of the species.

Ecological longevity of Polaskia chende (Cactaceae) seeds in the soil seed bank, seedling emergence and survival.

Soil seed banks are essential elements of plant population dynamics, enabling species to maintain genetic variability, withstand periods of adversity and persist over time, including for cactus species. However knowledge of the soil seed bank in cacti is scanty. In this study, over a 5-year period we studied the seed bank dynamics, seedling emergence and nurse plant facilitation of Polaskia chende, an endemic columnar cactus of central Mexico. P. chende seeds were collected for a wild population in Puebla, Mexico. Freshly collected seeds were sown at 25 °C and 12-h photoperiod under white light, far-red light and darkness. The collected seeds were divided in two lots, the first was stored in the laboratory and the second was use to bury seeds in open areas and beneath a shrub canopy. Seeds were exhumed periodically over 5 years. At the same time seeds were sown in open areas and beneath shrub canopies; seedling emergence and survival were recorded over different periods of time for 5 years. The species forms long-term persistent soil seed banks. The timing of seedling emergence via germination in the field was regulated by interaction between light, temperature and soil moisture. Seeds entered secondary dormancy at specific times according to the expression of environmental factors, demonstrating irregular dormancy cycling. Seedling survival of P. chende was improved under Acacia constricta nurse plants. Finally, plant facilitation affected the soil seed bank dynamics as it promoted the formation of a soil seed bank, but not its persistence.