Germination as an Unreliable Indicator of the Effectiveness of Cryopreservative Procedures for Imbibed Seeds

Tomato seeds of 49 per cent moisture content have been thawedfrom liquid nitrogen (—196 °C) with no loss of germinationfollowing both slow (12 °C min^–1) and rapid (850 °Cmin^–1) cooling, using 35 per cent dimethyl sulphoxide(DMSO) as a cryoprotectant. Lower concentrations of DMSO resultedin reduced germination. High proportions of seedlings from frozen/thawedseed failed to survive transplanting into soil, especially ifthey had been cooled rapidly. Seedlings grown to maturity afterthe slow cooling treatment showed a greater proportion of morphologicalabnormalities than a control population or those that had beenrapidly cooled. It is suggested that if cryopreservative techniques are to beevaluated for the storage of recalcitrant seeds, false impressionsof efficacy might be gained by reliance solely on germinationtesting. Cryopreservation, germination, recalcitrant seed     

A simple method for the freeze-preservation of zoospores of the green macroalga Enteromorpha intestinalis

Settled zoospores of the green macroalga Enteromorpha intestinalis were subjected to several different freezing and storing treatments at both cryogenic and non-cryogenic temperatures after which their viability was assessed using a spore germination bioassay. Three different cooling rates were tested: slow cooling at –1°C min⁻¹ and –0.5°C min⁻¹ to end temperatures in the range –20°C to –40°C, and a two-step procedure whereby the spores were frozen to –30°C at a rate of –1°C min⁻¹ prior to immersion in liquid nitrogen at –196°C. Spore viability was also investigated using the cryoprotectants glycerol and dimethyl suphoxide (DMSO), a reduced saline medium and various storage times. In the majority of experiments, the use of a cryoprotectant during the freezing process significantly increased the viability of the spores, with DMSO affording slightly greater protection than glycerol. All treatments produced high viabilities (ranging from 75.3–100.0%) after 5-min storage at the different end temperatures. However, progressively longer storage up to 7 days generally resulted in a marked reduction in viability. This was with the exception of spores frozen in a reduced saline medium; a medium of 75% seawater and either 5 or 10% DMSO greatly increased spore viability, with values of > 40% recorded for spores stored at –20°C for up to 5 weeks. This revised version was published online in July 2006 with corrections to the Cover Date.     

State and Phase Transition Behaviors ofQuercus rubraSeed Axes and Cotyledonary Tissues: Relevance to the Desiccation Sensitivity and Cryopreservation of Recalcitrant Seeds

Freezing and melting transitions of cellular water in embryonic axes and cotyledonary tissues of recalcitrantQuercus rubra(red oak) seeds were compared under slow and rapid cooling conditions. The relevance of desiccation sensitivity (critical water content) and state/phase transition behaviors to cryopreservation was examined. Under a slow to intermediate cooling condition (≤10°C min⁻¹), unfrozen water content in the tissues decreased to less than the critical water content, resulting in a dehydration damage. Under a rapid cooling condition (>100°C min⁻¹) using liquid nitrogen (LN₂), freeze-induced dehydration damage could be avoided if the initial water content was >0.50 g g⁻¹dry wt. However, at water content >0.50 g g⁻¹dry wt, the vitrified cellular matrix was highly unstable upon warming at 10°C min⁻¹. These results offered a theoretical explanation on the difficulty for successful cryopreservation of recalcitrant red oak embryonic axes. A complete state/phase transition diagram for red oak axes was constructed, and a vitrification-based cryopreservation protocol that employed predehydration and rapid cooling was examined. State/phase transition behaviors of cellular water are important parameters for cryopreservation; however, vitrification alone was not sufficient for seed tissues to survive the cryopreservation condition.     

Advances in the cryopreservation of sea-urchin embryos: Potential application in marine water quality assessment

Among the most widely used biological techniques in marine pollution assessment, the sea-urchin embryo–larval bioassay is in an advanced developmental stage. Cryopreservation might help to overcome the problem of the spawning seasonality and therefore strengthen the use of those embryo–larval bioassays. This work investigates different factors influencing cryopreservation of sea-urchin embryos, including the cooling rates and holding temperatures, the seeding, or the impact of plunging into liquid nitrogen. The blastula stage yielded better results than the fertilised egg, and the most effective cryoprotectants combination was dimethyl sulfoxide 1.5 M plus trehalose 0.04 M. The optimised protocol developed begins with an initial hold at 4 °C for 2 min, followed by cooling at −1 °C min⁻¹ to −12 °C. At this point a seeding step was incorporated with a 2 min hold, followed by a second cooling at −1 °C min⁻¹ to −80 °C. After a final hold of 2 min the cryovials are transferred into liquid nitrogen for storage. Although the cryopreservation processes might cause a delay in the development of sea-urchin embryos, high larval growth (71–98% of controls) was obtained when cryopreserved blastulae were further incubated for 72–96 h in artificial seawater. We conclude that embryo–larval bioassays with cryopreserved sea-urchin blastulae are suitable for use in marine pollution monitoring programs and may be considered as an acceptable solution to the reproductive seasonality of sea-urchin species.     

Towards integrated conservation of Australian endangered plants—the Western Australian model

Four percent of the Australian flora is rare and endangered with over 100 taxa presumed extinct. Western Australia contains a large proportion of the endangered flora of Australia with 238 taxa in a critical state of conservation and 70 species presumed extinct. Kings Park and Botanic Garden in south-west Australia is responsible for developing specialized collections of rare and endangered indigenous flora. Macro-and micropropagation procedures are used including conventional cutting and seed propagation, grafting and in thein vitro programme whole seeds (asymbiotic and symbiotic germination), excised seed embryos, shoot apices and inflorescence sections. Wherever possible explants are collected from major provenances of the species and a wide cross section of a species population. Although many of the rare flora of Western Australia are now in theex situ collection maintained by Kings Park and Botanic Garden attempts are being made to develop slow growth storage forin vitro cultures and cryostorage. Trial recovery programmes have commenced with a number of species including the rare and endangered Purdie's donkey orchid (Diuris purdiei). Results of these recovery programmes will guide future efforts in conserving and recovering rare Australian species.     

Seed banking of terrestrial orchids: evaluation of seed quality in Anacamptis following 4‐year dry storage

• Good‐quality dry seeds of some orchids have the potential to survive for decades under conventional seed bank conditions, but further research is needed to fill existing gaps in knowledge regarding seed behaviour under long‐term dry storage. The objectives of this study were to evaluate germination ability on two asymbiotic culture media with different nitrogen source; to assess seed desiccation tolerance needed for the storage at sub‐zero temperatures; and to study the effects of dry storage at low temperature.
• Asymbiotic seed germination tests of four Anacamptis species were carried out to evaluate the effects of different culture media, dehydration and dry storage on germination ability. Viability of 4‐year‐stored seeds was assessed by means of the tetrazolium test.
• Generalised linear model (GLM) analysis detected significant effects (P < 0.01) of the species, medium and storage time on total germination, while dehydration did not significantly affect it. Except for A. palustris, germination percentage was minimum after 1‐month storage and increased with longer storage periods. Tetrazolium viability tests detected high percentages of viable seed (>90%) following 4‐year storage in three out of four species.
• Seeds of the four Anacamptis species proved to be desiccation tolerant and have orthodox storage behaviour. The consequence of these findings is of interest to practical conservation approaches for orchids in seed‐banking. The results highlight the importance of multiple assessments of seed quality, both viability and germination, to understand seed storage behaviour.

     

Microalgal cryo-preservation using dimethyl sulfoxide (Me2SO) coupled with two freezing protocols: Influence on the fatty acid profile

Procedures for determining the optimal pre-freezing protocol for cryo-preservation of microalgae are discussed. Three algal species were used (Chlorella vulgaris, Isochrysis galbana and Dunaliella salina) and cryo-stored using two different methods: the slow cooling and the fast freezing. In the slow cooling, each algae batch was treated with or without cryo-protectant (dimethyl sulfoxide: Me₂SO 5% v/v). After 20 min at 4 °C, the midi-straws were filled and cooled slowly (1.5 °C min⁻¹) to −140 °C, by a programmable freezer (Digitcool—IMV), before putting them directly into liquid nitrogen. Fast freezing was performed with 10% or 15% Me₂SO prior to plunging into liquid nitrogen. The three algal species followed the same re-growth pattern as that of the controls. The post-thawed viability with Me₂SO was good for all the selected algae (C. vulgaris >95%, I. galbana and D. salina >70% of the control), applying the slow cooling. The post-thawed viability without Me₂SO was 60% for I. galbana, 52% for D. salina and 33% for C. vulgaris. Fast freezing was not suitable for cryo-storage of I. galbana but gave good post-thawing viability for D. salina (70%). The decrease in fatty acid content of the cryo-stored algae was influenced by the temperature. The rapid decrease in temperature induced by fast freezing can explain the low level of fatty acid content of the three cryo-stored algae. Fatty acid profiles show that the nutritional values of the three cryo-stored micro-algae were not significantly affected especially when treated with slow cooling protocols.     

The promotive effect of smoke derived from burnt native vegetation on seed germination of Western Australian plants

Exposure of dormant seed to cold smoke derived from burnt native vegetation had a positive influence on germination in one or more seed provenances in 45 out of 94 species of native Western Australian plants that are normally hard to germinate. When tested under controlled conditions some species showed earlier germination in smoke treatments than controls; in others smoke-treated seeds continued to germinate for several weeks after controls had achieved full germination. In the remainder, treated and control seeds germinated to similar time schedules. A group of 23 species which responded positively had previously been recorded as extremely difficult or impossible to germinate using conventional techniques. These included members of the genera Geleznowia (Rutaceae), Hibbertia (Dilleniaceae), Stirlingia (Proteaceae), Verticordia (Myrtaceae), Actinostrobus (Cupressaceae) and Pimelea (Thymelaeaceae). Both large- and small-seeded species were encountered amongst the positively responding taxa, which encompassed representatives of 15 families and 26 genera of dicotyledons, 5 families and 8 genera of monocotyledons and the gymnosperm Actinostrobus acuminatus. Sowing seeds on smoke-fumigated filter papers or watering with aqueous eluates of smoke elicited similar degrees of stimulation of germination, as did exposure to gaseous smoke in a readily germinating species Anigozanthos manglesii (Haemodoraceae) and the normally intractable species Lysinema ciliatum (Epacridaceae). Exposing recently burnt and unburnt natural bushland sites to smoke, smoked water or smoked dry sand elicited a significant germination response in 15 species. Over one third of the species sampled in the burnt site exhibited germination additional to that caused by the fire. Data are discussed in relation to previous germination studies on Australian and other taxa.     

Cryopreservation ofTetraselmis suecica cultured under different nutrients regimes

The commercially important member of the PrasinophyceaeTetraselmis suecica CCAP 66/4, which is widely used in the aquaculture industry, can be cryopreserved with levels of post-thaw viability in excess of 65%. The nutritional mode which the alga uses appears to be of secondary importance to the cryoprotectant and cooling protocol used, to maximise survival. Glycerol was the least cytotoxic and most protective of the cryoprotectants employed and a cooling rate of 5 °C min^-1 to -30 °C with a 10-minute equilibration, followed by plunging into liquid nitrogen was found to give the highest levels of post-thaw viability.     

Cryopreservation of periwinkle,Catharanthus roseus cells cultured in vitro

A procedure for prolonged cryogenic storage of periwinkle cell cultures is described. Cells derived from periwinkle, Catharanthus roseus (L.) G. Don, and subcultured as suspension in 1-B5C nutrient medium have been frozen, stored in liquid nitrogen (–196°C) for 11 weeks, thawed and recultured. Maximal survival was achieved when 3–4 day-old cells precultured for 24 h in nutrient medium with 5% DMSO were frozen at slow cooling rates of 0.5 or 1°C/min prior to storage in liquid nitrogen. The only loss in viability of cells occurred subsequent to treatment with DMSO. Abbreviations: DMSO, dimethylsulfoxide; 2,4-D, 2,4-dichlorophenoxyacetic acid; TTC, triphenyltetrazolium chloride.NRCC No. 20082     

Survival of somatic embryos and recovery of plants of sweet orange (Citrus sinensis (L.) Osb.) after immersion in liquid nitrogen

Somatic embryos of Washington Navel sweet orange (Citrus sinensis (L.) Osb.) derived from in vitro cultured ovules excised from immature fruits, were frozen to the temperature of liquid nitrogen. A method of slow cooling at a rate of 0.5°C min^-1 down to –42°C followed by storage in liquid nitrogen was used. Thawing was achieved by keeping the specimens at room temperature for 15 min. A small number of frozen embryos survived and developed into proliferating cultures that produced whole plants. The plants obtained from frozen cultures were transferred to soil and are growing successfully.     

Conservation technologies for safeguarding and restoring threatened flora: case studies from Eastern Australia

This paper highlights recent advances and improved scientific understanding of conservation technologies through selected case studies on threatened plant species indigenous to Eastern Australia. This includes investigations into seed desiccation, storage responses and cryopreservation in rainforest species, particularly the socio-economically important Australian native Citrus spp., Davidsonia spp. (Davidson’s plum) and Syzygium spp. This work also (1) increases our understanding of ecological correlates of seed desiccation sensitivity for predictive use and (2) improves restoration practice through better understanding of seed storage and germination requirements. The use of in vitro conservation technologies in support of conservation actions for endangered species is outlined in case studies on Wollemia nobilis (Wollemi pine), epiphytic and terrestrial orchid species, and an endangered fern species.     

Effects of temperature,light and gibberellic acid on the germination of seeds of 43 species native to Western Australia

Abstract. Species native to the southwest of Western Australia, representing a range of plant families, life-history strategies, fire-response syndromes, seed-store types and seed weights, were tested for viability using tetrazolium chloride and for germination under combinations of constant temperatures of 15 °C or 23 °C, constantly dark or 12 h diurnal whitelight conditions, and with, or without, addition of gibberellic acid (GA₃, 50 mg/l). Species previously known to require a heat-shock treatment to overcome dormancy due to an impervious testa were pre-treated prior to imposition of temperature, light and GA₃ conditions. The test environmental conditions related to differences between winter and autumn temperatures and surface and buried seed germination positions of post-fire habitats. The viability of the selection of native Western Australian species ranged from 0 to 100 %, averaging 71 %. For all taxa, no combination of temperature, light and gibberellic acid treatment induced all viable seeds to germinate. The greatest percentage germination achieved in any combination of treatments averaged 71 % of all viable seeds for all species. Larger seeds (> 10 mg seed weight) tended to have greater viability percentages, but no overall patterns of viability or germinability were attributable to seed storage syndrome, strategy of fire recovery response or life-form type. Germination of most of the dominant tree representatives (Eucalyptus calophylla, E. diversicolor, E. erythrocorys, E. gomphocephala, and E. patens) was indifferent to the trial conditions of temperature, light and GA₃. However, Eucalyptus marginata showed reduced germination in the light, which was overcome with GA₃. GA₃ also overcame the inhibition resulting from exposure to light in some understorey species (e.g. Allocasuarina campestris, Regelia ciliata, Xanthorrhoea gracilis and X. preissii). Germination of many hard-seeded, understorey shrub and herbaceous perennial species, especially those with small (< 10 mg) seeds (e.g. Bossiaea ornata, B. aquifolium and Acacia drummondii ssp. candolleana) was greater at the lower trial temperature and in the dark. Some large (> 10 mg) seeded, understorey species (e.g. Acacia extensa, Kennedia coccinea, K. prostrata, Hovea trisperma and Hardenbergia comptoniana) germinated in high percentages in both temperatures, but maximum germination percentages still tended to be at 15 °C. Large-seeded species were less sensitive to exposure to light compared to the smaller seeded species. The largest seeded species tested, Paraserianthes lophantha, germinated best in the warmer incubation temperature and in the light. The ecological significance of the tests would be that species which have seed dormancy mechanisms capable of delaying germination until the cool temperature, winter rainy period of this mediterranean-type climate would be more likely to survive than if germination followed summer rain showers or the first, intermittent rains of autumn. Burial of seeds becomes more important if germination occurs when rains first begin as this period has less available soil moisture and temperatures are high. Also survival of seedlings could be enhanced if germination of seed was restricted to the positions protected from high light, higher temperatures and lower soil moisture by the presence of a forest canopy. Therefore, seeds which have an ability to sense the presence of a previous fire in the habitat, conditions in light environment and appropriate temperature level have an adaptive advantage to time emergence to situations of time and space where survival is maximized. Variation in viability and germination percentages were apparent in some cases where more than one seed collection of available for testing, indicating that further aspects, such as seed age, maturity at collection, storage conditions and depth of seed dormancy, remain to be considered.     

Comparative cryopreservation study of trochophore larvae from two species of bivalves: Pacific oyster (Crassostrea gigas) and Blue mussel (Mytilus galloprovincialis)

Oysters and mussels are among the most farmed species in aquaculture industry around the world. The aim of this study was to test the toxicity of cryoprotective agents to trochophore larvae from two different species of bivalves and develop an improved cryopreservation protocol to ensure greater efficiency in the development of cryopreserved trochophores (14 h old oyster larvae and 20 h old mussel larvae) to normal D-larvae for future developments of hatchery spat production. The cryopreservation protocol producing the best results for oyster trochophores (60.0 ± 6.7% normal D-larvae) was obtained by holding at 0 °C for 5 min then cooling at 1 °C min⁻¹ to −10 °C and holding for 5 min before cooling at 0.5 °C to −35 °C, holding 5 min and then plunging into liquid nitrogen (LN), using 10% ethylene glycol. For mussel experiments, no significant differences were found when cooling at 0.5 °C min⁻¹ or at 1 °C min⁻¹ for CPA combinations with 10% ethylene glycol and at 0.5 °C min⁻¹. Using these combinations, around half of trochophores were able to develop to normal D-larvae post-thawing (48.9 ± 7.6% normal D-larvae).     

Seed germination ecology in southwestern Western Australia

Germination responses of species from the native plant communities of southwestern Western Australia can be related to syndromes of life history, fire response, and seed storage, and also to factors related to environmental stress. The Mediterranean-type climate of the region with periodic drought and recurrent fires affects the production of viable seeds in plants of limited stature and rooting depth. Fire response ephemerals and species cued to flower by fire tend to produce viable, readily germinable seeds, but there are instances where seed production is aborted in these predominantly herbaceous life forms. Clonal, rhizomatous species often produce mainly inviable seeds. Production of viable seeds in woody species of these highly diverse communities may also be restricted by limitations to cross pollination. Obligate post-fire seeding species tend to produce a greater proportion of viable seeds than species which are capable of resprouting following fire. Serotinous species, whether post-fire re-seeders or post-fire resprouting species, produce mainly viable seeds, which germinate readily once freed from protective fruits. Species of the legume families and a few others of the soil seed bank produce innately dormant seeds which can be germinated following heat shock treatments which simulate the effects of fire. Heat shock in these species appears mainly as a mechanism to crack the hard seed coats, but the effect of heat to denature seed coat inhibitors has not been eliminated. Western Australian species do not seem to break dormancy when exposed to leachates from burned wood as has been observed in comparable habitats in California and South Africa, but further research is advised. Germination in many native southwestern Australian species is cued by temperatures that correspond to the winter rainfall period. There are also indications that an after-ripening period of warm, dry storage increases percentage of germinable seeds. Stimulation of germination by hormones is almost unresearched in Western Australia, but germination percentages have been increased in a small number of species of horticultural potential. Stimulation of germination by soil nutrient concentrations is almost unresearched in Western Australia, except for the inhibitory effect of excess sodium chloride levels inEucalyptus andMelaleuca. These species only germinate when osmotic effects are reduced to lower levels as would occur when winter rains dilute soil salts. Application of research on seed germination has already enhanced the establishment of seedlings in the restoration of mine sites and is becoming important in aspects of the breeding and selection of native plants for the cut flower, bedding plant and essential oil industries.     

Cryopreservation of isolated micropores of spring rapeseed (Brassica napus L.) for in vitro embryo production

Microspore cryopreservation is a potentially powerful method for long-term storage of germplasm for in vitro embryo production in plant species. In this study, several factors influencing embryo production following the ultra-low temperature (–196 °C in liquid nitrogen) storage of isolated microspores of rapeseed (Brassica napus L.) were investigated. Microspores were prepared in cryogenic vials and subjected to various cooling treatments before immersion in liquid nitrogen for varying periods. Efficiency of microspore cryopreservation was reflected by in vitro embryo production from frozen microspores. Of all the cooling treatments, microspores treated with a cooling rate of 0.25% °C/min and a cooling terminal temperature of –35 °C before immersion in liquid nitrogen produced the highest embryo yields (18% and 40% of unfrozen controls in two genotypes, respectively). Fast thawing in a 35 °C water bath was necessary to recover a high number of embryos from microspore samples being frozen at a higher cooling rate, while thawing speed did not affect samples after freezing at a slower cooling rate. The storage density of cryopreserved microspores affected embryo production. Storage at the normal culture density (8×10⁴ microspores/ml) was less efficient for embryo production than at high densities (4×10⁶ microspores/ml and 1.6×10⁷ microspores/ml), although no significant difference was found between the high densities. Evaluation of plant lines derived from frozen microspores indicated no variation in isozyme pattern and no enhanced cold tolerance of these lines. Isolated microspores of B. napus could be stored for extended period for in vitro embryo production.     

Cryopreservation of Embryogenic Suspension Cultures of Barley (Hordeum vulgare L.)*

Anther-derived suspension cultures of Hordeun vulgare L. were cryopreserved by slow cooling and storage in liquid nitrogen for up to 55 days. Cells were pre-cultivated in L3 suspension medium supplemented with sorbitol. For freeze preservation the cells were treated with different combinations of cryoprotectant agents such as DMSO, proline, glycerol and sucrose. After rapid thawing high viabilities of up to 77% could be achieved. Cell growth commenced 2- 3 weeks later. The frequency of plantlet regeneration was 1%.     

Cryopreservation of wheat suspension culture and regenerable callus

Wheat (Triticum aestivum L. cv. Norstar) suspension cultures and regenerable calli initiated from immature embryos can be cryopreserved in liquid nitrogen temperature (–196°C) by slow freezing (0.5°C/min) in the presence of a mixture of DMSO and sucrose or sorbitol. Cold hardening or ABA treatment before cryopreservation increased the freezing resistance and improved the survival of wheat suspension culture in liquid nitrogen. Callus culture, established from immature embryos, prefrozen in 5% DMSO and 0.5M sorbitol survived liquid nitrogen storage and resumed plant regeneration after thawing. The results confirm the feasibility of long term preservation of wheat embryo callus by cryopreservation and retention of plant regeneration ability.Abbreviations ABA Abscisic acid - 2,4-D 2,4-Dichlorophenoxyacetic acid - DMSO Dimethylsulfoxide - LN Liquid nitrogen - TTC 2,3,5-triphenyltetrazolium chloride NRCC No. 23850.     

Comparison of heat transfer in liquid and slush nitrogen by numerical simulation of cooling rates for French straws used for sperm cryopreservation

Slush nitrogen (SN₂) is a mixture of solid nitrogen and liquid nitrogen, with an average temperature of −207 °C. To investigate whether plunging a French plastic straw (commonly used for sperm cryopreservation) in SN₂ substantially increases cooling rates with respect to liquid nitrogen (LN₂), a numerical simulation of the heat conduction equation with convective boundary condition was used to predict cooling rates. Calculations performed using heat transfer coefficients in the range of film boiling confirmed the main benefit of plunging a straw in slush over LN₂ did not arise from their temperature difference (−207 vs. −196 °C), but rather from an increase in the external heat transfer coefficient. Numerical simulations using high heat transfer (h) coefficients (assumed to prevail in SN₂) suggested that plunging in SN₂ would increase cooling rates of French straw. This increase of cooling rates was attributed to a less or null film boiling responsible for low heat transfer coefficients in liquid nitrogen when the straw is placed in the solid-liquid mixture or slush. In addition, predicted cooling rates of French straws in SN₂ tended to level-off for high h values, suggesting heat transfer was dictated by heat conduction within the liquid filled plastic straw.     

Optimising seed processing techniques to improve germination and sowability of native grasses for ecological restoration

• Grasslands across the globe are undergoing expansive degradation due to human impacts and climate change. If restoration of degraded native grassland is to be achieved at the scale now required, cost‐effective means for seed‐based establishment of grass species is crucial. However, grass seeds present numerous challenges associated with handling and germination performance that must be overcome to improve the efficiency of seeding. Previous research has demonstrated that complete removal of the palea and lemma (husk) maximises germination performance, hence we investigated the effects of complete husk removal on seed handling and germination of four temperate Australian grass species.
• Three techniques were tested to remove the husk – manual cleaning, flaming or acid digestion (the latter two followed by a manual cleaning step); these techniques were refined and adapted to the selected species, and germination responses were compared.
• The complete removal of the husk improved seed handling and sowability for all species. Germination was improved in Microlaena stipoides by 19% and in Rytidosperma geniculatum by 11%. Of the husk removal methods tested, flaming was detrimental to seed germination and fatal for one species (R. geniculatum). Compared to manual cleaning, sulphuric acid improved the overall efficacy of the cleaning procedure and increased germination speed (T50) in Austrostipa scabra, Chloris truncata and M. stipoides, and improved final germination in R. geniculatum by 13%.
• The seed processing methods developed and tested in the present study can be applied to grass species that present similar handling and germination performance impediments. These and other technological developments (seed coating and precision sowing) will facilitate more efficient grassland restoration at large scale.