Fiona R. Hay

Priming and re-drying improve the survival of mature seeds of Digitalis purpurea during storage

BACKGROUND AND AIMS Most priming studies have been conducted on commercial seed lots of unspecified uniformity and maturity, and subsequent seed longevity has been reported to both increase and decrease. Here a seed lot of Digitalis purpurea L. with relatively uniform maturity and known history was used to analyse the effects of priming on seed longevity in air-dry storage. METHODS Seeds collected close to natural dispersal and dried at 15% relative humidity (RH), 15 degrees C, were placed into experimental storage (60% RH, 45 degrees C) for 14 or 28 d, primed for 48 h at 0, -1, -2, -5, -10 or -15 MPa, re-equilibrated (47% RH, 20 degrees C) and then returned to storage. Further seed samples were primed for 2 or 48 h at -1 MPa and either dried at 15% RH, 15 degrees C or immediately re-equilibrated for experimental storage. Finally, some seeds were given up to three cycles of experimental storage and priming (48 h at -1 MPa). KEY RESULTS Priming at -1 MPa had a variable effect on subsequent survival during experimental storage. The shortest lived seeds in the control population showed slightly increased life spans; the longer lived seeds showed reduced life spans. In contrast, seeds first stored for 14 or 28 d before priming had substantially increased life spans. The increase tended to be greatest in the shortest lived fraction of the seed population. Both the period of rehydration and the subsequent drying conditions had significant effects on longevity. Interrupting air-dry storage with additional cycles of priming also increased longevity. CONCLUSIONS The extent of prior deterioration and the post-priming desiccation environment affect the benefits of priming to the subsequent survival of mature seeds. Rehydration-dehydration treatments may have potential as an adjunct or alternative to the regeneration of seed accessions maintained in gene banks for plant biodiversity conservation or plant breeding.

Habitat-correlated seed germination behaviour in populations of wood anemone ( Anemone nemorosa L.) from northern Italy

Although various aspects of the biology of Anemone nemorosa have been examined, few studies present data on seed germination, and even then information tends to be rather contradictory. A. nemorosa L. is a spring-flowering, woodland geophyte, widely distributed across much of Europe. Germination phenology, including embryo development and radicle and shoot emergence, were investigated in one mountain and three lowland populations from northern Italy. Immediately after harvest, seeds were either sown on agar in the laboratory under simulated seasonal temperatures, or placed in nylon mesh sachets and buried in the wild. Embryos, undifferentiated at the time of dispersal, grew under summer conditions in the laboratory and in the wild. However, seeds did not germinate under continuous summer conditions. Radicle emergence in the field was first recorded at the beginning of autumn, when soil temperatures had dropped to c. 15°C in the case of the three lowland populations, and to c. 10°C at the mountain site. Shoot emergence was delayed under natural conditions until late autumn/early winter, when soil temperatures had dropped to c. 10°C in the lowlands and c. 6°C at the mountain site. In the laboratory, a period of cold stratification was required for shoot emergence, and this requirement was more pronounced in the mountain population. Seeds of the mountain population completed embryo development, radicle emergence and shoot emergence at cooler temperatures compared with the lowland populations. These results suggest that germination in A. nemorosa is highly adapted and finely tuned to local climate. We conclude that seeds of A. nemorosa display deep, simple epicotyl, morphophysiogical dormancy, and this is the first report of such dormancy for the genus Anemone . However, the continuous development and growth of embryos from the time of natural dispersal, and the lack of evidence of developmental arrest under natural conditions, suggests that radicles are non-dormant.

Seed quality for conservation is critically affected by pre-storage factors

The quality of seed-conservation collections, and hence their value for species reintroduction or restoration, is critically dependent on factors operating in the period between the point of collection and arrival at environmentally controlled processing and storage facilities. The timing of the acquisition of desiccation tolerance and seed longevity in air-dry storage, in relation to mass maturity and the time of natural seed dispersal, varies across species. In some wild plant species, seed quality continues to improve up to, and possibly beyond, the point of dispersal. Holding immature berries of Solanum dulcamara L. and capsules of Digitalis purpurea L. under natural conditions enabled comparison of seed quality between seeds stored under natural conditions and those dried rapidly under seedbank dry-room conditions. While seeds from fully ripe (post-mature) capsules of D. purpurea were insensitive to different post-harvest drying treatments, seed quality declined when mature berries of S. dulcamara were held under natural conditions. These results emphasise that the selection of post-harvest treatment will not only depend on the maturity of collected seeds but also may vary across species depending on the fruit type. Except for subtropical and tropical coastal locations, ambient daytime conditions during the main seed-collecting season (November–February) across Australia can be expected to result in tolerable rates of seed deterioration for the duration of seed-collecting missions. However, because seed moisture levels can be considerably higher than when equilibrated with ambient relative humidity, post-harvest handling decisions should ideally be informed by measurements of seed moisture at the time of collection, and subsequently seed moisture should be monitored during transit.

Developmental changes in the germinability, desiccation tolerance, hardseededness, and longevity of individual seeds of Trifolium ambiguum.

BACKGROUND AND AIMS Using two parental clones of outcrossing Trifolium ambiguum as a potential model system, we examined how during seed development the maternal parent, number of seeds per pod, seed position within the pod, and pod position within the inflorescence influenced individual seed fresh weight, dry weight, water content, germinability, desiccation tolerance, hardseededness, and subsequent longevity of individual seeds. METHODS Near simultaneous, manual reciprocal crosses were carried out between clonal lines for two experiments. Infructescences were harvested at intervals during seed development. Each individual seed was weighed and then used to determine dry weight or one of the physiological behaviour traits. KEY RESULTS Whilst population mass maturity was reached at 33-36 days after pollination (DAP), seed-to-seed variation in maximum seed dry weight, when it was achieved, and when maturation drying commenced, was considerable. Individual seeds acquired germinability between 14 and 44 DAP, desiccation tolerance between 30 and 40 DAP, and the capability to become hardseeded between 30 and 47 DAP. The time for viability to fall to 50 % (p(50)) at 60 % relative humidity and 45 degrees C increased between 36 and 56 DAP, when the seed coats of most individuals had become dark orange, but declined thereafter. Individual seed f. wt at harvest did not correlate with air-dry storage survival period. Analysing survival data for cohorts of seeds reduced the standard deviation of the normal distribution of seed deaths in time, but no sub-population showed complete uniformity of survival period. CONCLUSIONS Variation in individual seed behaviours within a developing population is inherent and inevitable. In this outbreeder, there is significant variation in seed longevity which appears dependent on embryo genotype with little effect of maternal genotype or architectural factors.

The effect of maturity on the moisture relations of seed longevity in foxglove ( Digitalis purpurea L.)

Controlled aging experiments were carried out in order to evaluate the changes in seed longevity occurring following the attainment of maximum seed dry weight in foxglove (Digitalis purpurea L.). Seeds harvested in three successive years were stored at ~5% moisture content (fresh weight basis) and 50°C. Seed longevity was greater the later the seeds were harvested. When the data were modelled using the predictive viability model of Ellis and Roberts, these increases were attributable to increases in both the theoretical initial viability (in probits) of the seed-lot, Ki, and in the standard deviation of the normal distribution of seed deaths in time, σ. Furthermore, there was a positive relationship between Ki and σ which was independent of the year of harvest (r = 0.9016, for 11 d.f., P < 0.001). These results do not support the predictive model which relies on the assumption that σ will not differ between seed lots of the same species stored under identical conditions. When seeds were stored at a range of moisture contents (between 5 and 10%) at 50°C, increases in σ during seed development were manifest as changes in the negative logarithmic relationship between σ and moisture content; below an upper limit, the regressions for seed lots at different stages of maturity were significantly different (P < 0.05) but could be constrained to either a common intercept or to a common slope (P < 0.05). It is proposed that it was the inherent variation in individual seed lifespans which increased during seed development. These results raise important concerns regarding the use of predetermined ‘species constants’ to predict the longevity of seed lots during long-term storage in seed banks.

Ex situ conservation and cryopreservation of orchid germplasm

Premise of research. Orchids are among the most enigmatic of plant species. Yet the Orchidaceae comprises more species at risk of extinction than any other plant family. The collection and storage of orchid germplasm—principally seeds and associated mycorrhizal fungi but also protocorm-like bodies using encapsulation and vitrification techniques—allows for secure ex situ conservation. This article reviews the approaches and techniques used for the ex situ conservation of orchid germplasm, with a focus on seed banking and the use of cryopreservation techniques to improve the longevity of germplasm. Pivotal results. It is increasingly apparent that cryopreservation—the storage of germplasm at ultra-low temperatures (e.g., in liquid nitrogen)—is required for the long-term and low-maintenance conservation of all types of orchid germplasm. For orchid seeds, desiccation tolerance is common, but longevity in storage is poor. Cryopreservation of orchid seeds shows promise, but some complexities in low-temperature storage behavior still require explanation and resolution. The application of more advanced cryopreservation techniques, including encapsulation-dehydration and vitrification, is becoming increasingly common. These techniques provide for the simultaneous storage of orchid propagules with their compatible fungus, while for seeds, vitrification techniques show potential for improving tolerance to the stresses of cryopreservation. Conclusions. A renewed focus on describing the low-temperature storage physiology of orchid seeds to more precisely define the relationship between seed water content, storage temperature, and seed survival is required, as is perhaps the wider adoption of the use of cryoprotectants for seeds. This research, coupled with the development of improved methods of seed viability testing, will support the growing work of germplasm banks to protect orchid biodiversity in the face of habitat loss and potential species extinction.

Post-abscission, pre-dispersal seeds of Digitalis purpurea remain in a developmental state that is not terminated by desiccation ex planta

BACKGROUND AND AIMS Seed quality may be compromised if seeds are harvested before natural dispersal (shedding). It has been shown previously that slow or delayed drying can increase potential quality compared with immediate rapid drying. This study set out to investigate whether or not there is a critical moisture content, below which drying terminates maturation events for seeds harvested after mass maturity but before dispersal. METHODS Seeds of foxglove (Digitalis purpurea) in the post-abscission pre-dispersal phase were held at between 15 and 95 % RH for 4 or 8 d, with or without re-hydration to 95 % RH for a further 4 d, before drying to equilibrium at 15 % RH. In addition, dry seeds were primed for 48 h at -1 MPa. Subsequent seed longevity was assessed at 60 % RH and 45 degrees C. KEY RESULTS Rate of germination and longevity were improved by holding seeds at a wide range of humidities after harvest. Longevity was further improved by re-hydration at 95 % RH. Priming improved the longevity of the seeds dried immediately after harvest, but not of those first held at 95 % RH for 8 d prior to drying. CONCLUSIONS Maturation continued ex planta in these post-abscission, pre-dispersal seeds of D. purpurea dried at 15-80 % RH at a rate correlated positively with RH (cf. ageing of mature seeds). Subsequent re-hydration at 95 % RH enabled a further improvement in quality. Priming seeds initially stored air-dry for 3 months also allowed maturation events to resume. However, once individual seeds within the population had reached maximum longevity, priming had a negative impact on their subsequent survival.

Habitat-related germination behaviour and emergence phenology in the woodland geophyte Anemone ranunculoides L. (Ranunculaceae) from northern Italy

This study examined whether the restricted habitat preference of the spring-flowering woodland geophyte Anemone ranunculoides L., compared with that of A. nemorosa growing in the same woodlands in northern Italy, could be explained by subtle differences in germination preference and emergence phenology. Immediately after harvest, seeds of A. ranunculoides were either sown on agar in the laboratory under simulated seasonal temperatures or placed in nylon mesh sachets and buried in the wild. Embryos, undifferentiated at the time of seed dispersal, grew during summer in the laboratory and in the wild, culminating in radicle emergence in the autumn, when temperatures fell to c. 15°C. Shoot emergence was delayed under natural conditions until soil temperature had dropped further to c. 10°C. Compared with populations of the closely related Anemone nemorosa L. occupying the same woodland habitat, which have been reported to have non-dormant radicles, A. ranunculoides displayed a narrower temperature tolerance for radicle emergence and high levels of germination were possible only after prolonged exposure to summer conditions, indicating physiological dormancy. However, unlike A. nemorosa , shoot emergence in A. ranunculoides was not dependent on winter temperatures, suggesting weaker epicotyl morphophysiological dormancy. Under a regime of diurnal temperature alternation, simulating the microclimate where there is little plant cover, germination failed almost completely; this could explain the absence of A. ranunculoides in open habitats.

Temperature control of seed germination in Fritillaria tubiformis subsp. moggridgei ( Liliaceae ) a rare endemic of the South-west Alps

Fritillaria tubiformis subsp. moggridgei (Liliaceae) is a rare, endemic species that inhabits open mountains and alpine grasslands of the Ligurian Alps. At the time of seed dispersal, the underdeveloped embryos were 27% the length of the seed. Here we report the results of laboratory experiments carried out to determine the temperature preferences for embryo growth and radicle emergence. Embryo growth commenced immediately after sowing at 4°C. Once the embryo had grown the length of the seed, the radicle emerged. The time required for embryo growth and radicle emergence was longer when seeds were placed through a seasonal sequence of temperatures, commencing with late summer (10/20°C), compared with seeds immediately placed at a temperature to simulate winter conditions (4°C). Prematurely transferring seeds from winter to spring temperatures (5/10°C) also slowed the progress of germination. Radicle emergence did not occur at 10 or − 5°C and less than 20% germination occurred in seeds placed at constant 0°C. Addition of gibberellic acid (GA3) did not promote embryo growth of seeds placed at 20°C. Overall, the temperature preferences for embryo growth and subsequent radicle emergence are such that, in situ, seed germination may occur during the winter under snow cover or at the end of winter to coincide with snow melt and warming temperatures.

Comparative longevity and low-temperature storage of seeds of Hydatellaceae and temporary pool species of south-west Australia

The comparative longevity of seeds of species from the early-angiosperm group, Hydatellaceae, along with other temporary wetland aquatics from the South-west Australian Floristic Region were tested under standard experimental storage conditions. In contrast to recent hypotheses proposing that seeds from basal angiosperm species may be short-lived in storage, seeds of the Hydatellaceae species (Trithuria submersa Hook.f. and T. austinensis D.D.Sokoloff, Remizowa, T.Macfarlane and Rudall) were longer-lived than the other temporary wetland aquatic species tested. Seeds of Glossostigma drummondii Benth. (Scrophulariaceae), Myriophyllum petreaum Orchard and M. balladoniense Orchard (Haloragaceae), lost viability quickly and are thus predicted to be short-lived in seed bank storage. To assist seed bank conservation programs, the effect of seed moisture content on the viability of seeds stored for 1, 6 and 12 months at −18°C or in vapour phase cryopreservation (−150°C) was determined. Seeds of all species survived storage at both temperatures for up to 12 months, provided seed equilibrium relative humidity was below ~50%. Given the high conservation value of Hydatellaceae species and the potential short-lived nature of seeds of some of the species, we recommend that ex situ conservation programs for these aquatic species should consider cryopreservation as a means to maximise the longevity of their seeds.