Hugh W. Pritchard

The science and economics of ex situ plant conservation

Ex situ seed storage underpins global agriculture and food supplies and enables the conservation of thousands of wild species of plants within national and international facilities. As an insurance policy against extinction, ex situ seed conservation is estimated to cost as little as 1% of in situ conservation. The assumptions, costs, risks and scientific challenges associated with ex situ plant conservation depend on the species, the methods employed and the desired storage time. Recent, relatively widespread evidence of less than expected longevity at conventional seed bank temperatures, innovations in the cryopreservation of recalcitrant-seeded species and economic comparators provide compelling evidence that ultra-cold storage should be adopted for the long-term conservation of plants. Policy instruments, such as the Global Strategy for Plant Conservation (2011-2020), should respond to the evidence base and promote the implementation of cryopreservation for both tropical and temperate plants.

Oleosins prevent oil-body coalescence during seed imbibition as suggested by a low-temperature scanning electron microscope study of desiccation-tolerant and -sensitive oilseeds

Abstract. In order to clarify further the physiological role of oleosins in seed development, we characterized the oil-body proteins of several oilseeds exhibiting a range of desiccation sensitivities from the recalcitrant (Theobroma cacao L., Quercus rubra L.), intermediate (Coffea arabica L., Azadirachta indica A. Juss.) and orthodox categories (Sterculia setigera Del., Brassica napus L.). The estimated ratio of putative oleosins to lipid in oil bodies of Q. rubra was less than 5% of the equivalent values for rapeseed oil bodies. No oleosin was detected in T. cacao oil bodies. In A. indica cotyledons, oil bodies contained very low amounts of putative oleosins. Oil bodies both from C. arabica and S. setigera exhibited a similar ratio of putative oleosins to lipid as found in rapeseed. In C. arabica seeds, the central domain of an oleosin was partially sequenced. Using a low temperature field-emission scanning electron microscope, the structural stability of oil bodies was investigated in seeds after drying, storage in cold conditions and rehydration. Despite the absence or relative dearth of oleosins in desiccation-sensitive, recalcitrant oilseeds, oil bodies remained relatively stable after slow or fast drying. In A. indica seeds exposed to a lethal cold storage treatment, no significant change in oil-body sizes was observed. In contrast, during imbibition of artificially dried seeds containing low amounts of putative oleosins, the oil bodies fused to form large droplets, resulting in the loss of cellular integrity. No damage to the oil bodies occurred in imbibed seeds of Q. rubra, C. arabica and S. setigera. Thus the rehydration phase appears to be detrimental to the stability of oil bodies when these are present in large amounts and are lacking oleosins. We therefore suggest that one of the functions of oleosins in oilseed development may be to stabilize oil bodies during seed imbibition prior to germination.

Butenolide from plant-derived smoke enhances germination and seedling growth of arable weed species

We tested the applicability of the recently identified major germination cue from smoke (a butenolide 3-methyl-2Hfuro[2,3-c]pyran-2-one) on 18 weed species from non-fire prone environments. For the study species we compared the relative effectiveness of alternating temperatures, KNO3, GA3, smoke water and the butenolide on germination percentage, germination rate and seedling mass. We found that while smoke stimulated germination in a number of species it also had negative impacts on other species. In addition, the butenolide was effective on the widest range of species in terms of enhancing germination percentage, rate and seedling mass. However, none of the treatments, including butenolide were effective on all species. Our data demonstrate that butenolide may have wide applicability as a germination and seedling growth stimulant irrespective of whether the species come from fire-prone habitats.

Ecological correlates of seed desiccation tolerance in tropical African dryland trees

In the tropics, species with recalcitrant or desiccation-sensitive, Type III seeds are largely restricted to regions with comparatively high rainfall, because desiccation-induced seed death will be minimal in these environments. However, species with recalcitrant seeds do occur in drylands, although little is known about ecological adaptations to minimize seed death in these environments. Here we present data for the seed desiccation tolerance of 10 African dryland species and examine the relationships between seed size, rainfall at the time of seed shed, and desiccation tolerance for these and a further 70 species from the scientific literature. The combined data set encompasses species from 33 families. Three species (Syzygium cumini, Trichilia emetica, and Vitellaria paradoxa) had desiccation-sensitive seeds, and the remaining seven species investigated were desiccation-tolerant. The desiccation-sensitive species had large (>0.5 g) seeds, germinated rapidly, and had comparatively small investments in seed physical defenses. Furthermore, seed was shed in months of high rainfall (>60 mm). In comparison, for species with desiccation-tolerant seeds, seed mass varied across five orders of magnitude, and seed was shed in wet and dry months. Although infrequent in dryland environments (approximately 11% of the species examined here), species with desiccation-sensitive seeds do occur; large size, rapid germination, and the timing of dispersal all reduce the likelihood of seed drying. Furthermore, desiccation-sensitivity may be advantageous for large-seeded species by increasing the efficiency of resource use in seed provisioning.

Variable desiccation tolerance in Acer pseudoplatanus seeds in relation to developmental conditions: a case of phenotypic recalcitrance?

Nine seedlots of the widely planted southern and central European native tree species Acer pseudoplatanus L. were collected along a north-south gradient spanning 21° of latitude in Europe. We investigated how the heat sum during seed development influences seed maturity as assessed by physical, physiological and biochemical traits. Using principal component analysis we found predictable and consistent patterns in all traits, which correlated with heat sum. For example, compared with fruits from their native range (Italy and France, heat sum >3000°C d), fruits from the coldest location (Scotland; heat sum of 1873°C d) were shorter (c. 30 v. 42 mm), germinated over a narrower temperature range (5-20 v. 5-35°C) and had smaller embryos (28 v. > 70 mg) with a higher water content (c. 63 v. 48%), less negative solute potentials (c. -2.4 v. -4.1 MPa) and were more desiccation sensitive (critical water potential of -20.2 v. -55.4 to -60.7 MPa). The observed level of desiccation-tolerance for the French and Italian seedlots is more consistent with the intermediate category than the previous classification of A. pseudoplatanus as recalcitrant. Our results demonstrate that a lower heat sum causes fruits from northern Europe to be dispersed before maximum potential seed quality is achieved.

Development of a thermal time model for the quantification of dormancy loss in Aesculus hippocastanum seeds

The effects of temperature on dormancy loss, germination and viability were investigated in seeds of Aesculus hippocastanum L. harvested over a 4-year period. Release from embryo dormancy in freshly harvested seeds was manifest in two phases of morphological growth: initially, when the seed lot was only partially released, axis emergence resulted primarily from cotyledonary petiole extension without radicle extension; subsequently, when the seed lot was totally released, axis emergence of all seeds was followed immediately by extension to >1 cm through growth of the radicle. Germination (axis emergence and radicle extension) at 16°C was a function of pre-treatment period at 2–11°C. The rate of dormancy loss (probit germination d −1 ) increased linearly below a ceiling temperature for the chilling response; this temperature was estimated to vary from 13°C to 16°C for two seed lots harvested in separate years. Dormancy periods for individual seeds within both seed lot populations can be described by cumulative normal distributions; the predicted standard deviation of chilling units below the ceiling temperature (i.e. thermal time) was 186°C d. Visible germination occurred during the process of stratification at 2°C, starting after 21–25 weeks. By contrast, three years of hy-drated seed storage at 16°C, which was a non-permissive temperature for dormancy loss, resulted in little pre-emergence of the axis during stratification; approximately one third of the seeds remained germinable. The implications of these quantitative analyses of the physiological processes in recalcitrant seeds for the development of improved storage methods are discussed.

Ex Situ Conservation of Orchids in a Warming World

Whilst there is overwhelming scientific evidence that dramatic changes in regional climates are likely to occur throughout the 21st century, the scientific community remains uncertain how the effects of global heating will combine with other environmental factors to affect wild orchid populations. It is, however, likely that many populations will be affected adversely and that in situ conservation techniques by themselves will not be sufficient to prevent the extinction of many species. A range of complimentary ex situ strategies are discussed. Amongst these orchid seed banking has been shown to be an invaluable tool for conserving the maximum amount of genetic diversity in the minimum space and has the potential to enable the conservation of valuable material for possible re-introduction and habitat restoration programmes in the future. The Darwin Initiative project, ‘Orchid Seed Stores for Sustainable Use’ (OSSSU), is currently establishing a global network of orchid seed banks focussing initially on countries with high orchid biodiversity in Asia and Latin America. Particular reference is made to ex situ conservation in China, together with the urgent need to gather more data to determine which habitats and species are most at risk of extinction in the wild in the immediate future.

Noninvasive diagnosis of seed viability using infrared thermography

Recent advances in the noninvasive analyses of plant metabolism include stress imaging techniques, mainly developed for vegetative tissues. We explored if infrared thermography can be used to predict whether a quiescent seed will germinate or die upon water uptake. Thermal profiles of viable, aged, and dead Pisum sativum seeds were recorded, and image analysis of 22,000 images per individual seed showed that infrared thermography can detect imbibition- and germination-associated biophysical and biochemical changes. These “thermal fingerprints” vary with viability in this species and in Triticum aestivum and Brassica napus seeds. Thermogenesis of the small individual B. napus seeds was at the limit of the technology. We developed a computer model of “virtual pea seeds,” that uses Monte Carlo simulation, based on the heat production of major seed storage compounds to unravel physico-chemical processes of thermogenesis. The simulation suggests that the cooling that dominates the early thermal profiles results from the dissolution of low molecular-weight carbohydrates. Moreover, the kinetics of the production of such “cooling” compounds over the following 100 h is dependent on seed viability. We also developed a deterministic tool that predicts in the first 3 hours of water uptake, when seeds can be redried and stored again, whether or not a pea seed will germinate. We believe that the early separation of individual, ungerminated seeds (live, aged, or dead) before destructive germination assessment creates unique opportunities for integrative studies on cell death, differentiation, and development.

Desiccation-induced dormancy in papaya ( Carica papaya L.) seeds is alleviated by heat shock

The effects of desiccation and temperature on the germination capability of Carica papaya L. were investigated for seeds extracted from three commercial fruit batches. More than 50% of freshly isolated, cleaned (sarcotesta removed) but undried seeds germinated at 26°C. However, desiccation to approx. 20%seed RH reduced germination at this temperature to

Inter-nucleosomal DNA fragmentation and loss of RNA integrity during seed ageing

The germination of viable seeds is the basis for new plant growth and development. Seeds lose viability during storage, but the biochemical mechanisms of seed death are not fully understood. This study aimed to investigate degradation patterns of nucleic acids during seed ageing and subsequent water uptake. Seeds of Pisum sativum L. were artificially aged at 50°C and 12% seed water content (WC). Nucleic acids degradation was studied during ageing and during imbibition of four seed lots with differential viability from highly viable to dead. As seeds lost viability during ageing, DNA was gradually degraded into internucleosomal fragments, resulting in ‘DNA laddering’, in conjunction with disintegration of 18S and 28S rRNA bands. During imbibition, non-aged controls had high levels of DNA and RNA integrity through to radicle protrusion. In an aged seed lot with 85% total germination (TG) DNA fragmentation decreased upon imbibition probably due to nucleosome degradation, while rRNA integrity did not improve. In an aged seed lot with 44% TG, neither DNA nor rRNA integrity improved upon imbibition. Dead seeds showed DNA degradation as laddering throughout imbibition along with extensive degradation of rRNA. We present a model in which interlinked programmed and non-programmed events contribute to seed ageing, and suggest that protection of nucleic acids during ageing is key to seed longevity.