Optimal hydration status for cryopreservation of intermediate oily seeds: Citrus as a case study

BACKGROUND AND AIMS: The purpose of this study was to investigate the basis of the optimal hydration status for cryopreservation of intermediate oily seeds using Citrus as a model. METHODS: The relationships between equilibrium relative humidity (RH), seed water content, presence of freezable water as determined by DSC analysis, and germination percentage after immersion in liquid nitrogen (LN) were investigated in Citrus aurantifolia, C. grandis, C. madurensis and C. reticulata. The relationship between the lipid content of seeds and their unfrozen water content was also investigated. KEY RESULTS: Independent of their level of seed desiccation tolerance, the optimal desiccation RH for seed tolerance to LN exposure was 75-80 % in the four species studied. This optimal hydration status always coincided with that at which presence of frozen water could not be detected in seed tissues during the cooling/thawing process. The unfrozen water content of seeds was variable between species and negatively correlated to seed lipid content. Using the present data, those obtained previously in seven coffee species and those reported by other authors for five other species, a significant linear relationship was found between the lipid content and the unfrozen water content of seeds. CONCLUSIONS: This study provides additional evidence that intermediate oily seeds do not withstand the presence of freezable water in their tissues during the cooling/warming process. Moreover, it offers two important applied perspectives: (1) independent of their level of desiccation tolerance, testing germination of seeds of a given oily seed species after equilibration in 75-80 % RH at 25 degrees C and LN exposure, gives a rapid and reliable evaluation of the possibility of cryopreserving whole seeds of this given species; (2) it is now possible to calculate the interval of water contents in which non-orthodox oily seeds of a given species are likely to withstand LN exposure as a function of their lipid content.     

生菜种子低温处理后耐冻性和脂肪酸合成代谢的关系研究

以生菜(Lactuca sativa)种子为研究对象,通过不同时间的吸水处理分析其含水量变化,再通过程序降温处理,分析不同含水量种子发芽率的差异,以及脂肪酸合成有关基因(FAD2、FAD3、PPT、ELOVL)和冷调节基因ICE1的表达。结果表明,种子含水量随吸水时间增加而升高。程序降温至同样的低温冷冻条件下(-20℃、-22℃),吸水时间小于6 h的种子发芽率较高,而吸水8 h以上的种子发芽率显著降低。种子吸水8 h含水量处于饱和状态,在此状态下种子对低温较为敏感,说明含水量对种子耐冻性有影响。冷冻处理后生菜种子基因表达检测结果表明,脂肪酸去饱和酶基因(FAD2、FAD3)、蛋白质棕榈酰基硫脂酶相关基因(PPT）、长链脂肪酸延伸酶相关基因(ELOVL)的表达水平均随着种子含水量增加呈上升趋势,吸胀10 h的种子表达量最高,此时种子由于高含水量所受冷冻伤害最大。基因ICE1在冷冻处理种子中的表达也随着吸水时间增加而升高,在吸水10 h时种子中表达量到最高水平。综上,种子含水量越高,所受冷冻伤害越大。但种子在低温条件下具有一定的抗冷反应,可通过相关基因的过表达调控合成更多不饱和脂肪酸、抗冻蛋白等提高含水种子耐冻性。     

Cold tolerance of a New Zealand alpine cockroach, Celatoblatta quinquemaculata (Dictyoptera, Blattidae)

Abstract. Ecophysiological features, including survival and recovery from freezing and determination of the freezable water content, are reported for a cold-adapted cockroach Celatoblatta quinquemaculata Johns 1966 (Dictyoptera, Blattidae) inhabiting alpine communities at altitudes greater than 1300 m a.s.l. in mountains of Central Otago, New Zealand. Nymphs ranged from 15 to 51 mg live weight of which 67% was water. Cockroaches had a mean supercooling point temperature of ?5.4 ± 0.1°C; with recovery from freezing close to this temperature being rapid, but no recovery was observed when frozen at ?9 to ?10°C. The duration of exposure to freezing conditions and the time allowed for recovery (24–96 h) both influenced individual recovery and subsequent survival. Comparison of supercooling point data and survival shows that this species possesses a few degrees of freeze tolerance, and individuals have been found frozen in the field when subzero temperatures occur. Differential scanning calorimetry showed ≈ 74% of body water froze during cooling and between 24 and 27% of total body water was osmotically inactive (unfreezable under the experimental conditions). Carbohydrates, other than glucose at 7.5μg/mg fresh weight, were in low concentrations in the body fluids, suggesting little cryoprotection. No thermal hysteresis from antifreeze protein activity was detected in haemolymph samples using calorimetric techniques. It is suggested that slow environmental cooling rates, together with high individual supercooling points, confer a small amount of freezing tolerance on this species enabling it to survive low winter temperatures. This has allowed it to colonize and maintain populations in alpine habitats > 1300 m a.s.1. in New Zealand.     

Basis of coffee seed sensitivity to liquid nitrogen exposure: oxidative stress or imbibitional damage?

Two hypotheses, namely the occurrence of post‐thaw oxidative stress or imbibitional damage, were tested to explain the high sensitivity of coffee seeds to liquid nitrogen (LN) exposure. Oxidative stress was studied by measuring primary and secondary products of lipid peroxidation in seeds during the desiccation and rehydration periods. The 4‐hydroxynonenal (4‐HNE) content of seeds remained constant throughout the desiccation step. No significant difference was observed between desiccated seeds and seeds desiccated and exposed to LN for the evolution of their 4‐HNE and hydroperoxide contents during rehydration. In both cases, an increase in 4‐HNE and hydroperoxide contents of seeds was observed during the first hours of culture under germination conditions, followed by a progressive decrease down to values comparable to those observed in desiccated seeds. The hydroperoxide composition of frozen seeds was not significantly different from that of control seeds. The (S)/(R) enantiomeric ratios of 9‐ and 13‐hydroxy‐octadecadienoic acid extracted from rehydrating seeds were chiral, suggesting that they originated from lipoxygenase activity. These results suggest that the high sensitivity of coffee seeds to LN exposure is not directly associated with the occurrence of an oxidative stress during post‐thaw rehydration. The effect on seed viability of different rehydration procedures previously identified to reduce membrane imbibitional injury was studied after desiccation and LN exposure. Desiccation tolerance increased with, by increasing order, seed osmoconditioning, pre‐heating and pre‐humidifying prior to their culture under germination conditions. Among the four combinations of pre‐humidification durations (24 or 48 h) and temperatures (25 or 37°C) tested, pre‐humidification for 24 h at 37°C gave the highest level of desiccation tolerance. This rehydration procedure also dramatically increased seed viability after LN exposure. Seed desiccation sensitivity modelling in combination with the calculation of the decrease in seed water activity during cooling facilitated the explanation of the beneficial effect of controlled rehydration after desiccation and LN exposure. These results support the hypothesis that imbibitional membrane damage is involved in the sensitivity of coffee seeds to LN exposure.     

Freezing and desiccation tolerance of imbibed canola seed

Depending on the environmental conditions, imbibed seeds survive subzero temperatures either by supercooling or by tolerating freezing-induced desiccation. We investigated what the predominant survival mechanism is in freezing canola ( Brassica napus cv. Quest) and concluded that it depends on the cooling rate. Seeds cooled at 3°C h⁻¹ or faster supercooled, whereas seeds cooled over a 4-day period to −12°C and then cooled at 3°C h⁻¹ to−40°C did not display low temperature exotherms. Both differential thermal analysis and nuclear magnetic resonance (NMR) spectroscopy confirmed that imbibed canola seeds undergo freezing-induced desiccation at slow cooling rates. The freezing tolerance of imbibed canola seed (LT₅₀) was determined by slowly cooling to −12°C for 48 h, followed with cooling at 3°C h⁻¹ to −40°C, or by holding at a constant −6°C (LD₅₀). For both tests, the loss in freezing tolerance of imbibed seeds was a function of time and temperature of imbibition. Freezing tolerance was rapidly lost after radicle emergence. Seeds imbibed in 100 μ M abscisic acid (ABA), particularly at 2°C, lost freezing tolerance at a slower rate compared with water-imbibed seeds. Seeds imbibed in water either at 23°C for 16 h, or 8°C for 6 days, or 2°C for 6 days were not germinable after storage at −6°C for 10 days. Seeds imbibed in ABA at 23°C for 24 h, or 8°C for 8 days, or 2°C for 15 days were highly germinable after 40 days at a constant −6°C. Desiccation injury induced at a high temperature (60°C), as with injury induced by freezing, was found to be a function of imbibition temperature and time.     

Physiological aspects of Taxus brevifolia seeds in relation to seed storage characteristics

Water relations, desiccation tolerance and longevity of Taxus brevifolia (Nutt.) seeds were studied to determine the optimal stage of development and storage conditions for seeds of this species. Seeds equilibrated to a range of relative humidities (RHs) had unusually low water contents which can be accounted for by the high lipid content of gametophyte tissues (71% of the dry mass). Water relations of embryonic tissue were more typical of those reported for other seed species. The water content below which freezing transitions were not observable in the embryo was ca 0.24 g H₂O (g dry weight)⁻¹ (g g⁻¹) for all maturity classes studied. Embryos did not achieve significant levels of desiccation tolerance (survival to water contents less than 0.5 g g⁻¹) until the latter stages of development when dry matter was maximal. Mature embryos could be dried to 0.025 g g⁻¹ (seed water content of 0.010 g g⁻¹) with no loss of viability. Thus, at the latter stages of development, embryo water content could be optimized to avoid both desiccation and freezing damage. Survival of mature seeds declined over a 2-year period when seeds were stored at temperatures between 5 and 35°C and RHs between 14 and 75%, corresponding to seed water contents between 0.015 and 0.07 g g⁻¹. The deterioration rate was slowest for seeds stored at the lowest RH and temperature. Our data indicate that seeds of Taxus brevifolia show orthodox rather than recalcitrant storage characteristics, but that the optimum water content for storage was extremely low. The results suggest that even if stored at optimal water contents and low temperatures, T. brevifolia seeds will be relatively short lived. The high quantity of lipids or reducing sugars may be contributing factors in the poor storage characteristics.     

Temporal release of fatty acids and sugars in the spermosphere: impacts on Enterobacter cloacae-induced biological control

The aim of this study was to determine the temporal release of fatty acids and sugars from corn and cucumber seeds during the early stages of seed germination in order to establish whether sugars found in exudate can prevent exudate fatty acid degradation by Enterobacter cloacae. Both saturated (long-chain saturated fatty acids [LCSFA]) and unsaturated (long-chain unsaturated fatty acids [LCUFA]) fatty acids were detected in corn and cucumber seed exudates within 15 min after seed sowing. LCSFA and LCUFA were released at a rate of 26.1 and 6.44 ng/min/seed by corn and cucumber seeds, respectively. The unsaturated portion of the total fatty acid pool from both plant species contained primarily oleic and linoleic acids, and these fatty acids were released at a combined rate of 6.6 and 0.67 ng/min/seed from corn and cucumber, respectively. In the absence of seed exudate sugars, E. cloacae degraded linoleic acid at rates of 29 to 39 ng/min, exceeding the rate of total fatty acid release from seeds. Sugars constituted a significant percentage of corn seed exudate, accounting for 41% of the total dry seed weight. Only 5% of cucumber seed exudate was comprised of sugars. Glucose, fructose, and sucrose were the most abundant sugars present in seed exudate from both plant species. Corn seeds released a total of 137 microg/seed of these three sugars within 30 min of sowing, whereas cucumber seeds released 0.83 microg/seed within the same time frame. Levels of glucose, fructose, and sucrose found in corn seed exudate (90 to 342 microg) reduced the rate of linoleic acid degradation by E. cloacae to 7.5 to 8.8 ng/min in the presence of either sugar, leaving sufficient concentrations of linoleic acid to activate Pythium ultimum sporangia Our results demonstrate that elevated levels of sugars in the corn spermosphere can prevent the degradation of LCUFA by E. cloacae, leading to its failure to suppress P. ultimum sporangial activation, germination, and subsequent disease development.     

束花石斛种子超低温保存的研究

以濒危兰科物种野生束花石斛（Dendrobium chrysanthum）的成熟种子为材料,研究了PVS2植物玻璃化液对其萌发的作用,以及快速冷冻法和玻璃化法对种子超低温保存的影响。结果表明,种子的萌发率随着PVS2处理时间的延长而下降。PVS2预处理能明显地增加种子的超低温耐性。当预处理时间为15～45min时,种子的超低温耐性随预处理时间的延长而增加;当预处理时间长于60min后,种子的超低温耐性随预处理时间的延长而下降。经液氮保存后,存活的种子能萌发成为正常的幼苗。结论是经PVS2预处理45min后,成熟的束花石斛种子能成功地进行超低温保存。     

Winterfat (Eurotia lanata(Pursh) Moq.) Seedbed Ecology: Low Temperature Exotherms and Cold Hardiness in Hydrated Seeds as Influenced by Imbibition Temperature

Thermal analyses of freezing events in hydrated lettuce (LactucasativaL.) seeds show a correlation between low temperature exotherms(LTEs) (evidence of ice crystal formation) and seed death. Yet,weather patterns common to the Northern Great Plains of NorthAmerica regularly create conditions where non-dormant seedsof native plants hydrate with snow melt and are subsequentlyexposed to -30 °C or colder conditions. To determine ifsuch weather patterns decimate dispersed seeds, we measuredthe effects of freezing on fully hydrated winterfat (Eurotialanata(Pursh) Moq.) seeds harvested from the Northern Plainsat two USA and one Canadian location. Survival of hydrated seedsto -30 °C at a cooling rate of 2.5 °C h^-1was similarto that of seeds not subjected to cooling, even though botha high temperature exotherm (HTE) and an LTE were observed.Although the LTE was not related to winterfat seed survival,freeze-stressed seeds had reduced germination rates and reducedseedling vigour, particularly for the collection with the lightestseeds. The temperature of LTEs was similar among seed collectionswith a mean of -17.6 °C, but was warmer when the seeds wereimbibed at 0 °C compared to 5, 10 or 20 °C. We founda significant correlation between the HTE and LTE temperatures.The difference and the correlation may be due to the highermoisture content of seeds imbibed at 0 °C. After pericarpremoval, only one exotherm in the range of the LTE was observed.This was also true for the naked embryo. We conclude that anLTE indicates ice formation in the embryo, but that it doesnot signal the death of a winterfat seed.Copyright 1998 Annalsof Botany Company Eurotia lanata(Pursh) Moq.,Krascheninnikovia, Ceratoides,winterfat, exotherm, freezing tolerance, freezing avoidance, seedbed ecology, germination, D50, seedling vigour, seed collection     

Maize seed cryo-storage modifies chlorophyll,carotenoid, protein,aldehyde and phenolics levels during early stages of germination

We recorded the crypreservation effects (direct immersion) on various parameters of early germination stages of maize seeds (0, 7 and 14 days). Percentages of germination; fresh mass of different seedling parts; levels of chlorophyll pigments (a, b); carotenoids; malondialdehyde; other aldehydes; phenolics (cell wall-linked, free) and proteins were determined. Various statistically significant effects of seed exposure to liquid nitrogen (LN) were recorded. Maize seeds did not seem to be affected by LN exposure either visually or regarding fresh weight or germination rate. However, delayed growth was observed in seedlings recovered from cryopreserved seeds. This trend indicated an increase in the effect of seed cryopreservation on growing plants. The most significant effects of LN exposure were recorded in the combined fresh weight of stems and leaves at day 7 of germination and in fresh weights of roots, stems and leaves at day 14. At the biochemical level, numerous indicators varied following LN exposure, but the most significant effects were recorded in carotenoids, malondialdehyde and other aldehyde contents. LN exposure modified 50.0% of indicators in cotyledons, 48.1% in stems and leaves, 38.8% in roots and 11.1% in seeds. LN storage modified 11.1% of the variables measured at day 0 of germination, 37.0% at day 7, and 52.7% at day 14. Field performance of cryostored seed-derived plants should be evaluated to measure the durability of the changes observed.     

The influence of the rate of temperature change on the activation of dormant seeds of Rumex obtusifolius L.

1. One temperature shift from 20 to 30°C in darkness induces 30–40% germination in Rumex obtusifolius seeds. The same germination percentages are found with heat treatment varying between 1 and 6h duration, indicating that the total heat sum of the temperature shift is not important.
2. Germination is greatly enhanced by three consecutive heat shifts of 1h at 30°C separated by 1h periods at 20°C.
3. The seeds are activated to a small extent after a slow warming (+2°Ch^–1) from 20 to 30°C, followed by incubation for 1h at 30°C. Germination is much higher after rapid heating (+10°Ch^–1) to 30°C, followed by 1h incubation at this temperature. Repeated fast heating treatments on four consecutive days enhances germination. Moderately rapid heatings (+3·3°Ch^–1) give intermediate results.
4. The rate of cooling does not influence the germination percentage. Cooling alone cannot induce germination.
5. Heating alone from 15 to 25°C without cooling also activates germination. In this temperature range the seeds are more activated by rapid warming than by slow warming.
6. The ecological relevance of the response to different warming rate is discussed. The insensitivity of seeds to a slow warming might keep deeply buried seeds in a dormant stage.     

The effects of soil metals on the composition of oil of Paeonia ostii seeds

Peony seed oil has been known to have a high value for health due to the high content of unsaturated fatty acid. However, few studies have investigated which factors impact the oil quality and composition of seeds in the traditional mining cultivation area. In the present study, the investigation was conducted in 30 cultural field sites. Significantly negative relationships were found between Cu content in soils or leaves and linoleic and unsaturated fatty acids. Further tests revealed that Cu evidently accumulated in leaves. This demonstrated Cu content in soils was the important factor that influenced linoleic acid content and unsaturated fatty acid content. Such proposition must be considered when cultivating groups of plant toward expanding oil production.     

Effect of cooling and rewarming rates on glycerolated human erythrocytes.

Human erythrocytes suspended in a sodium-free buffered salt solution containing glycerol in 1 m concentration (1 part of packed cells to 4 parts buffered salt solution) were frozen by slow, moderately rapid, or very rapid cooling to various subzero C temperatures. The frozen specimens, after a 5-min storage period at a given temperature, were thawed at low, moderately high, or very high rates. The hemolysis in the frozen and thawed samples was measured by a colorimetric determination of the hemoglobin released from the damaged cells. At ?10 °C, the highest freezing temperature employed, nearly 100% recovery of intact erythrocytes was obtained irrespective of the cooling and rewarming conditions. The extent of the hemolysis after exposure to lower freezing temperatures depended upon the cooling and rewarming conditions. Moderately rapid and very rapid freezing to, and thawing from temperatures below ?40 °C permitted significantly higher recoveries of intact cells than the other freezing/ thawing combinations. In the temperature range ?15 to ?30 °C the combination slow cooling and slow rewarming afforded maximum protection. Very rapid freezing/ slow thawing was the most damaging combination throughout the entire freezing range. The results were interpreted in part by a conventional two-factor analysis, lower cooling rates allowing concentrated salts to determine hemolysis, higher cooling rates destroying the cells by intracellular freezing. Apparent anomalies were explained in terms of a generalized “thermal/osmotic” shock according to which the erythrocytes were subject to greater hemolysis the higher the rates of cooling and/or warming.     

Warming and elevated CO2 affect the relationship between seed mass, germinability and seedling growth in Austrodanthonia caespitosa, a dominant Australian grass

While the influence of elevated CO₂ on the production, mass and quality of plant seeds has been well studied, the effect of warming on these characters is largely unknown; and there is practically no information on possible interactions between warming and elevated CO₂, despite the importance of these characters in population maintenance and recovery. Here, we present the impacts of elevated CO₂ and warming, both in isolation and combination, on seed production, mass, quality, germination success and subsequent seedling growth of Austrodanthonia caespitosa , a dominant temperate C₃ grass from Australia, using seeds collected from the TasFACE experiment. Mean seed production and mass were not significantly affected by either elevated CO₂ or warming, but elevated CO₂ more than doubled the proportion of very light, inviable seeds ( P < 0.05) and halved mean seed N concentration ( P < 0.04) and N content ( P < 0.03). The dependence of seed germination success on seed mass was affected by an elevated CO₂× warming interaction ( P < 0.004), such that maternal exposure to elevated CO₂ or warming reduced germination if applied in isolation, but not when applied in combination. Maternal effects were retained when seedlings were grown in a common environment for 6 weeks, with seedlings descended from warmed plants 20% smaller ( P < 0.008) with a higher root : shoot ratio ( P < 0.001) than those from unwarmed plants. Given that both elevated CO₂ and warming reduced seed mass, quality, germinability or seedling growth, it is likely that global change will reduce population growth or distribution of this dominant species.     

On the similarity in the effects of different cryopreservation conditions on the growth and development of plants

The morphological characteristics of 35 wild plant species were studied after freezing of seeds under the conditions of deep, fast, and programmed freezing (-196 degrees C) and non-deep freezing (-10 degrees C). The seeds were stored frozen for a month. The seeds of all the species were characterized by a low humidity. The field and laboratory seed germination capacity, leaf growth, the quantity and length of shoots, the quantity of generative organs, and the variability of these characteristics were studied. It was shown that the direction of changes under different cooling conditions was the same except for the laboratory germination capacity of some species. The direction was determined by the species features rather than cooling conditions.     

Reactive oxygen species release, vitamin E, fatty acid and phytosterol contents of artificially aged radish (Raphanus sativus L.) seeds during germination

Seeds of Raphanus sativus L. subjected to accelerated ageing were investigated for reactive oxygen species (ROS) release and for content of vitamin E (tocopherol, TOC, and tocotrienol, TOC-3), fatty acids and phytosterols in seed coats, cotyledons and embryonic axes during germination. In unaged seeds, ROS release occurred mainly in seed coats of non-imbibed seeds and in seedlings (48?h of imbibition). TOC and TOC-3 were mainly represented by the ??-isoform, abundant in embryonic axes. Fatty acids were mainly found in cotyledons. In seed coat and embryonic axis, phytosterols consisted mainly of sitosterols. The effects of ageing were mainly visible in embryonic axes at 48?h of imbibition. Deterioration was associated with a decrease in fresh weight increase percentage, germination percentage, ??-TOC and total fatty acid content. An increase in ROS release from seed coats and in ??-TOC, ??-TOC, ??-TOC-3 content in embryonic axis was also observed. The use of ??-TOC and total fatty acids in embryonic axis as parameters of seed quality evaluation during storage was suggested.     

花楸树种子散布、萌发与种群天然更新的关系

花楸树是我国东北林区重要的非木质资源树种,其实生天然更新不良.本文通过研究花楸树种子散布、土壤种子库及种子萌发出土过程,分析花楸树实生天然更新的影响因素.结果表明：自然散落的花楸树果实96.1%分布于母株2 m范围内,凋落物层和土壤表层（0～2 cm）的种子数占土壤种子库总数的97.0%;不同季节花楸树土壤种子库种子数量差别很大,当年11月上旬种子数量最多,达(257.7±69.2)粒·m^-2;翌年7月下旬种子数量最少,仅为(2.9±2.9)粒·m^-2;温度不是花楸树种子萌发出土过程的限制因子,0 ℃～5 ℃时幼苗出苗率达(67.5±6.6)%,但对其出苗速率影响显著.土壤含水量为50%时,花楸树出苗率最高,达(74.7±4.2)%;含水量为60%时,幼苗死亡率最低,为(32.6±0.6)%.花楸树种子的散布格局和土壤种子库的时空分布格局影响种子的萌发出土过程,进而影响其种群的天然更新.     

Lipids in Berseem Seed (Trifolium alexandrinum) during Germination as Affected by Gibberellic Acid and Maleic Hydrazide

Lipid depletion of berseem seed treated with 100 mg/1 gibberellic acid or maleic hydrazide was studied during germination. During the first 3 days after germination there was little lipid consumption but thereafter the depletion of lipid was rapid. Gibberellic acid was found to enhance the lipid utilization of total lipids and triglyceride components during germination. On the other hand, maleic hydrazide reduced the utilization of triglycerides of the germinating seed. The rate of utilization was rapid for unsaturated fatty acids and slower for saturated ones in treated seeds as compared to untreated seeds.     

Characteristics and composition of Parkia biglobbossa and Jatropha curcas oils and cakes

Parkia biglobbossa (PKBS) and Jatropha curcas (JTC) seeds were analysed for their proximate composition. The seeds oils were analysed for fatty acid, lipid classes, sterols and physicochemical characteristics. Proximate analysis revealed that the percentage crude protein, crude fat and moisture in PKBS were 32.40%, 26.525% and 10.18% respectively and 24.60%, 47.25% and 5.54% in JTC. Campesterol, stigmasterol, beta-sitosterol, Delta5-avenasterol and Delta7-stigmasterol were identified in the seed oils, but beta-sitosterol was most abundant, constituting 71.9% in JTC and 39.5% in PKBS. JTC oil had 72.7% unsaturated fatty acids with oleic acid predominating, while PKBS had 62% unsaturated fatty acids with linoleic acid being the most abundant. Results of lipid classes showed triglyceride as the dominant lipid species in the seed oils. Physicochemical analysis of the seed oils showed that they could be classified as semi drying oils and that they could be found applicable in alkyd resin and soap manufacture.     

Germination and emergence of annual species and burial depth: Implications for restoration ecology

Due to the high content of viable seeds, topsoil is usually spread on ground left bare during railway and motorway construction to facilitate the regeneration of vegetation cover. However, during handling of the topsoil, seeds are often buried deeply and they cannot germinate or the seedlings cannot emerge from depth. This study experimentally explores the predictive value of seed mass for seed germination, mortality and seedling emergence at different burial depths for 13 common annual species in semiarid Mediterranean environments. We separate the effect of burial depth on germination and emergence by means of two experiments. In the germination experiment, five replicates of 20 seeds for each species were buried at depths ranging from 0 to 4 cm under greenhouse conditions. Germinated and empty or rotten seeds were counted after 8 weeks. In the emergence experiment, five replicates of four newly-germinated seeds per species were buried at the same depths under controlled conditions and emergence was recorded after 3 weeks. The effect of burial depth on percentage of germination and seedling emergence was dependent on seed size. Although all species showed a decrease in germination with burial depth, this decrease was greater for small-than large-seeded species. Percentage of emergence was positively related to seed mass but negatively related to burial depth. Seed mortality was higher for small-than large-seeded species, but there was no general effect of burial depth on this variable. Thus, the current practice of spreading 30 cm deep layers of topsoil in post-construction restoration projects is unadvisable. In this restoration scenario, thinner layers of topsoil should be used to achieve the maximum potential of the topsoil for germination and seedling establishment.