Monitoring of oxidative status in three native Australian species during cold acclimation and cryopreservation.

KEY MESSAGE: Three wild species exhibited a significant reduction in antioxidants throughout the cryopreservation protocol, whilst the half-cell reduction potential became more oxidised. Antioxidant content recuperated in recovering shoot tips. Cryopreservation is the most efficient and cost-effective long-term storage solution for the conservation of a wide range of plant species and material. Changes in the levels of antioxidants during the process of cryopreservation are known to reduce post-cryogenic survival due to oxidative stress. Low-molecular-weight thiols (cysteine, γ-glutamylcysteine, and glutathione) and ascorbic acid, which represent the two major water-soluble antioxidants in plants, were analysed at specific stages during cryopreservation of shoot tip material of three native Australian plant species [Anigozanthos viridis (Haemodoraceae), Lomandra sonderi (Asparagaceae), and Loxocarya cinerea (Restionaceae)] to quantify the oxidative stress experienced during cryopreservation. Post-cryogenic regeneration of shoot tips was greatest in A. viridis (78%) followed by L. sonderi (50%), whilst L. cinerea did not show any post-cryogenic regeneration. The application of a 3-week cold (5 °C) preconditioning regime, commonly used to increase post-cryogenic survival, resulted in significantly lower post-cryogenic regeneration for A. viridis (33%), but had little effect on the other two species. Total antioxidant concentration in shoot material decreased significantly with each step throughout the cryopreservation process, particularly in the cryoprotection and washing stages. Antioxidant levels in shoot tips then increased during the subsequent 7-day post-cryopreservation recovery period, with the greatest increase measured in A. viridis. Concentrations of thiols and their corresponding disulphides were used to calculate the corresponding half-cell reduction potentials, whereby the ability of these plant species to maintain a strong reducing environment in shoot tissues throughout the cryopreservation protocol was found to correlate with post-cryogenic survival.

Seed dormancy and persistent sediment seed banks of ephemeral freshwater rock pools in the Australian monsoon tropics.

BACKGROUND AND AIMS: Rock pools are small, geologically stable freshwater ecosystems that are both hydrologically and biologically isolated. They harbour high levels of plant endemism and experience environmental unpredictability driven by the presence of water over variable temporal scales. This study examined the hypothesis that the sediment seed bank in monsoon tropical freshwater rock pools would persist through one or more periods of desiccation, with seed dormancy regulating germination timing in response to rock pool inundation and drying events. METHODS: Seeds were collected from seven dominant rock pool species, and germination biology and seed dormancy were assessed under laboratory conditions in response to light, temperature and germination stimulators (gibberellic acid, karrikinolide and ethylene). Field surveys of seedling emergence from freshwater rock pools in the Kimberley region of Western Australia were undertaken, and sediment samples were collected from 41 vegetated rock pools. Seedling emergence and seed bank persistence in response to multiple wetting and drying cycles were determined. KEY RESULTS: The sediment seed bank of individual rock pools was large (13 824 ± 307 to 218 320 ± 42 412 seeds m(-2) for the five species investigated) and spatially variable. Seedling density for these same species in the field ranged from 13 696 to 87 232 seedlings m(-2). Seeds of rock pool taxa were physiologically dormant, with germination promoted by after-ripening and exposure to ethylene or karrikinolide. Patterns of seedling emergence varied between species and were finely tuned to seasonal temperature and moisture conditions, with the proportions of emergent seedlings differing between species through multiple inundation events. A viable seed bank persisted after ten consecutive laboratory inundation events, and seeds retained viability in dry sediments for at least 3 years. CONCLUSIONS: The persistent seed bank in freshwater rock pools is likely to provide resilience to plant communities against environmental stochasticity. Since rock pool communities are often comprised of highly specialized endemic and range-restricted species, sediment seed banks may represent significant drivers of species persistence and diversification in these ecosystems.

Evaluation of the new vacuum infiltration vitrification (viv) cryopreservation technique for native Australian plant shoot tips.

BACKGROUND: The application of a vacuum during the incubation in cryoprotective agents such as PVS2 allows for increased penetration, reducing total incubation times required before vitrification and post-cryopreservation regeneration is achieved. OBJECTIVE: This study compared a conventional droplet-vitrification protocol to the new vacuum infiltration vitrification protocol in four Australian plant species. MATERIALS AND METHODS: The new vacuum infiltration vitrification applied an 80 kPa vacuum during incubations in loading solution and PVS2. Infiltration of the cryoprotective agents into shoot tips was determined by differential scanning calorimetry measuring ice formation in the thermographs comparing a range of loading solution and PVS2 incubation times. RESULTS AND CONCLUSION: The application of the vacuum infiltration vitrification technique resulted in a significantly reduced PVS2 incubation time for cryogenic survival and regeneration for all four species, reducing the time needed to adequately protect shoot tips by half to a quarter when compared to a conventional droplet-vitrification technique.

Same, same, but different: dissimilarities in the hydrothermal germination performance of range-restricted endemics emerge despite microclimatic similarities.

Seed germination responses for most narrow-range endemic species are poorly understood, imperilling their conservation management in the face of warming and drying terrestrial ecosystems. We quantified the realized microclimatic niches and the hydrothermal germination thresholds in four threatened taxa (Tetratheca erubescens, Tetratheca harperi, Tetratheca paynterae subsp. paynterae and Tetratheca aphylla subsp. aphylla) that are restricted to individual Banded Ironstone Formations in Western Australia. While T. aphylla subsp. aphylla largely failed to germinate in our trials, all other species demonstrated extended hydrothermal time accumulation (186-500°C MPa days), cool minimum temperatures (7.8-8.5°C), but broad base water potential thresholds (-2.46 to -5.41 MPa) under which germination occurred. These slow germination dynamics are suggestive of cool and wet winter months, where soil moisture is retained to a greater capacity in local microsites where these species occur, rather than the warmer and drier conditions in the surrounding arid environment. Hydrothermal time-to-event modelling showed that each species occupied unique hydrothermal germination niches, which correspond with the microclimatic differences the species are exposed to. Our results provide a baseline understanding for environmental and germination thresholds that govern the recruitment, and ultimately the population structure and persistence, of these short-range endemic plants. In addition, our results can aid future conservation, as well as restoration actions such as translocation to bolster population numbers and to mitigate against losses due to anthropogenic disturbance and global environmental change.

Development of cryopreservation for Loxocarya cinerea---an endemic Australian plant species important for post-mining restoration.

We report the development of a cryopreservation protocol for the endemic Western Australian plant species Loxocarya cinerea (Restionaceae). Shoot tips from two genotypes, SXH404 and SXH804, were cryopreserved using the droplet-vitrification technique. Control explants, which were cryoprotected, but not cooled, showed regeneration for both genotypes (SXH404, 22.1 +/- 5.9%; SXH804, 67.7 +/- 9.6%). Extension of incubation in PVS2 from 30 to 60 min did not lead to survival after cryopreservation. Thermal analysis using differential scanning calorimetry confirmed the beneficial effect of a loading phase but also revealed no or very little ice formation after cryoprotection of shoot tips in other treatments. Regeneration following cryopreservation was obtained for genotype SXH804 (4.3 +/- 2.1%) but not for SXH404. Regenerated explants of L. cinerea SXH804 were morphologically identical to tissue-cultured plants. As an alternative to shoot tips, callus tissues of clone SXH404 were successfully cryopreserved (> 66.7% post LN survival) using the same protocol.

Are current seed storage approaches suitable for Macrozamia fraseri (Cycadales), a temperate species used in restoration?

In this study, we focused on understanding key storage traits of seeds from Macrozamia fraseri, an unusual though important species that is impacted by mining. To support current restoration activities, large amounts of seed from M. fraseri have been regularly collected and stored for up to 8 years under standard seed banking conditions (5°C and 20% relative humidity), though in situ recruitment from directly sown seed is poor. To investigate the underlying constraints to germination on demand, we set out to assess the viability of M. fraseri seeds that had been stored in a restoration seed bank from 6 to 66 months. Seed moisture content (MC) (fresh weight basis) was also determined for seeds with different storage histories to ascertain whether M. fraseri seeds display traits (i.e. high MC) that might suggest non-orthodox seed storage behaviour. The youngest seed accession (6 months old) was found to have a high MC (45.8 ± 5.4%-fresh weight basis), and >50% viability. In comparison, older (>30 months old) accessions were observed to have a marked reduction in both seed MC (10-35% MC) and viability (0-29.4%). While preliminary, we conclude that M. fraseri seeds appear to lose viability during conventional storage with younger accessions displaying both a higher seed MC and viability, compared to accessions stored for longer. Given the significance of these results, future research activities are recommended to better understand the interplay between seed MC and storage environment and how this relates to the seasonally dry Mediterranean climate where this species naturally occurs. As well, storage and propagation approaches are proposed to increase success when using M. fraseri for conservation and restorative activities.

Sympatric species of Hibbertia (Dilleniaceae) vary in dormancy break and germination requirements: implications for classifying morphophysiological dormancy in Mediterranean biomes.

BACKGROUND AND AIMS: Several ecologically important plant families in Mediterranean biomes have seeds with morphophysiological dormancy (MPD) but have been poorly studied. The aim of this study was to understand the seed ecology of these species by focusing on the prominent, yet intractably dormant Australian genus Hibbertia. It was hypothesized that the slow germination in species of this genus is caused by a requirement for embryo growth inside the seed before germination, and that initiation of embryo growth is reliant upon a complex sequence of environmental cues including seasonal fluctuations in temperature and moisture, and an interplay with light and smoke. Using the results, the classification of the MPD level in species of Hibbertia is considered. METHODS: Four species of Hibbertia in winter rainfall south-western Australia were selected. These species, whilst differing in geographic distributions, are variously sympatric, and all are important understorey components of plant communities. The following aspects related to dormancy break, embryo growth and germination were investigated: temperature and moisture requirements; effects of karrikinolide, gibberellic acid and aerosol smoke; and phenology. KEY RESULTS: Following exposure to wet/dry cycles at low or high temperatures, embryo growth and germination occurred, albeit slowly in all species at low temperatures when moisture was unlimited, corresponding to winter in south-west Australia. Photo regime influenced germination only in H. racemosa. Aerosol smoke triggered substantial germination during the 1st germination season in H. huegelii and H. hypericoides. CONCLUSIONS: Although the study species are con-generic, sympatric and produce seeds of identical morphology, they possessed different dormancy-break and germination requirements. The physiological component of MPD was non-deep in H. racemosa but varied in the other three species where more deeply dormant seeds required >1 summer to overcome dormancy and, thus, germination was spread over time. Embryos grew during winter, but future studies need to resolve the role of cold versus warm stratification by using constant temperature regimes. To include Mediterranean species with MPD, some modifications to the current seed-dormancy classification system may need consideration: (a) wet/dry conditions for warm stratification and (b) a relatively long period for warm stratification. These outcomes have important implications for improving experimental approaches to resolve the effective use of broadcast seed for ecological restoration.

Prior hydration of Brassica tournefortii seeds reduces the stimulatory effect of karrikinolide on germination and increases seed sensitivity to abscisic acid.

BACKGROUND AND AIMS: The smoke-derived compound karrikinolide (KAR(1)) shows significant potential as a trigger for the synchronous germination of seeds in a variety of plant-management contexts, from weed seeds in paddocks, to native seeds when restoring degraded lands. Understanding how KAR(1) interacts with seed physiology is a necessary precursor to the development of the compound as an efficient and effective management tool. This study tested the ability of KAR(1) to stimulate germination of seeds of the global agronomic weed Brassica tournefortii, at different hydration states, to gain insight into how the timing of KAR(1) applications in the field should be managed relative to rain events. METHODS: Seeds of B. tournefortii were brought to five different hydration states [equilibrated at 15 % relative humidity (RH), 47 % RH, 96 % RH, fully imbibed, or re-dried to 15 % RH following maximum imbibition] then exposed to 1 nm or 1 microm KAR(1) for one of five durations (3 min, 1 h, 24 h, 14 d or no exposure). KEY RESULTS: Dry seeds with no history of imbibition were the most sensitive to KAR(1); sensitivity was lower in seeds that were fully imbibed or fully imbibed then re-dried. In addition, reduced sensitivity to KAR(1) was associated with an increased sensitivity to exogenously applied abscisic acid (ABA). CONCLUSIONS: Seed water content and history of imbibition were found to significantly influence whether seeds germinate in response to KAR(1). To optimize the germination response of seeds, KAR(1) should be applied to dry seeds, when sensitivity to ABA is minimized.

High-resolution distribution modeling of a threatened short-range endemic plant informed by edaphic factors.

Short-range endemic plants often have edaphic specializations that, with their restricted distributions, expose them to increased risk of anthropogenic extinction.Here, we present a modeling approach to understand habitat suitability for Ricinocarpos brevis R.J.F.Hend. & Mollemans (Euphorbiaceae), a threatened shrub confined to three isolated populations in the semi-arid south-west of Western Australia. The model is a maximum entropy species distribution projection constructed on the basis of physical soil characteristics and geomorphology data at approximately 25 m2 (1 arc-second) resolution.The model predicts the species to occur on shallow, low bulk density soils that are located high in the landscape. The model shows high affinity (72.1% average likelihood of occurrence) for the known populations of R. brevis, as well as identifying likely locations that are not currently known to support the species. There was a strong relationship between the likelihood of R. brevis occurrence and soil moisture content that the model estimated at a depth of 20 cm.We advocate that our approach should be standardized using publicly available data to generate testable hypotheses for the distribution and conservation management of short-range endemic plant species for all of continental Australia.

Hydrological and thermal responses of seeds from four co-occurring tree species from southwest Western Australia.

Seed germination is a critical stage in the life cycle of most plants and is defined by specific tolerance thresholds beyond which rates and success of germination rapidly decline. Previous studies have demonstrated that widespread plant species commonly germinate over a broad range of temperatures and water stress levels, whereas range-restricted species often exhibit a narrower germination window in terms of temperature and moisture. We investigated the relationships of the key germination traits of maximum germination (G max) and time to 50% germination (t 50) in response to temperature (5-35°C) and water stress (-1.5-0 MPa) in four co-occurring Western Australian native Eucalyptus species with widely varying biogeography. Eucalyptus caesia subsp. caesia and E. ornata exhibit a highly localized distribution and a narrow geographical range, being restricted either to granite outcrops or the upper slopes and tops of lateritic rises, respectively. These two species were compared with the two widespread and dominant congenerics E. salmonophloia and E. salubris. There was a distinctive hump-shaped response of t 50 to temperature and an exponential response to water stress, characteristic of rate- and threshold-limited processes, but no consistent pattern in the response of G max. The four species were significantly different in their thermal performance of t 50, with E. caesia and E. ornata displaying narrower thermal tolerance ranges than the two widespread species. In terms of mean final germination percentage, the two range-restricted endemic taxa exhibited higher lability in their response to thermal stress and drought stress compared to the two broadly distributed congenerics. These findings indicate a link between distributional extent, temperature and water stress tolerance and may have implications for identifying ecological filters of rarity and endemism.

Defining the role of fire in alleviating seed dormancy in a rare Mediterranean endemic subshrub.

Fire is a topical issue in the management of many ecosystems globally that face a drying climate. Understanding the role of fire in such ecosystems is critical to inform appropriate management practices, particularly in the case of rare and ecologically specialized species. The Mediterranean heathlands are highly fire-prone and occur in a biodiversity hotspot increasingly threatened by human activities, and determining the reproductive thresholds of at-risk heathland species is critical to ensuring the success of future conservation initiatives. This study examined the germination biology of the threatened carnivorous subshrub Drosophyllum lusitanicum, with specific focus on the role of fire-related cues (heat and smoke) in combination with seasonal temperatures and moisture conditions to determine how these factors regulate seed dormancy and germination. We found that D. lusitanicum produces water-permeable, physiologically dormant seeds with a fully developed, capitate embryo that when fresh (~1 month old) and without treatment germinate to 20-40 % within 4-8 weeks. Seeds possess a restricted thermal window (15-20 °C) for germination and a neutral photoblastic response. Seed dormancy was overcome through precision nicking of the seed coat (>90 % germination) or by short exposure to dry heat (80 or 100 °C) for 5-30 min (60-100 % germination). We propose seedling emergence from the soil seed bank may be cued by the passage of fire, or by soil disturbance from the movement and browsing of animals. Long-term population viability is likely to be contingent upon appropriate management of the persistent soil seed bank, as well as the adequate management of key ecological disturbances such as fire. Drosophyllum lusitanicum faces an increasingly bleak future in the absence of conservation and management initiatives aimed at reducing habitat fragmentation in heathlands and aligning fire management and livestock practices with biodiversity outcomes.

Using hyperspectral imaging to determine germination of native Australian plant seeds.

We investigated the ability to accurately and non-destructively determine the germination of three native Australian tree species, Acacia cowleana Tate (Fabaceae), Banksia prionotes L.F. (Proteaceae), and Corymbia calophylla (Lindl.) K.D. Hill & L.A.S. Johnson (Myrtaceae) based on hyperspectral imaging data. While similar studies have been conducted on agricultural and horticultural seeds, we are unaware of any published studies involving reflectance-based assessments of the germination of tree seeds. Hyperspectral imaging data (110 narrow spectral bands from 423.6nm to 878.9nm) were acquired of individual seeds after 0, 1, 2, 5, 10, 20, 30, and 50days of standardized rapid ageing. At each time point, seeds were subjected to hyperspectral imaging to obtain reflectance profiles from individual seeds. A standard germination test was performed, and we predicted that loss of germination was associated with a significant change in seed coat reflectance profiles. Forward linear discriminant analysis (LDA) was used to select the 10 spectral bands with the highest contribution to classifications of the three species. In all species, germination decreased from over 90% to below 20% in about 10-30days of experimental ageing. P50 values (equal to 50% germination) for each species were 19.3 (A. cowleana), 7.0 (B. prionotes) and 22.9 (C. calophylla) days. Based on independent validation of classifications of hyperspectral imaging data, we found that germination of Acacia and Corymbia seeds could be classified with over 85% accuracy, while it was about 80% for Banksia seeds. The selected spectral bands in each LDA-based classification were located near known pigment peaks involved in photosynthesis and/or near spectral bands used in published indices to predict chlorophyll or nitrogen content in leaves. The results suggested that seed germination may be successfully classified (predicted) based on reflectance in narrow spectral bands associated with the primary metabolism function and performance of plants.

Little evidence for fire-adapted plant traits in Mediterranean climate regions.

As climate change increases vegetation combustibility, humans are impacted by wildfires through loss of lives and property, leading to an increased emphasis on prescribed burning practices to reduce hazards. A key and pervading concept accepted by most environmental managers is that combustible ecosystems have traditionally burnt because plants are fire adapted. In this opinion article, we explore the concept of plant traits adapted to fire in Mediterranean climates. In the light of major threats to biodiversity conservation, we recommend caution in deliberately increasing fire frequencies if ecosystem degradation and plant extinctions are to be averted as a result of the practice.