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Consequence of polystyrene particles size and concentration in plant tissues contamination.

With the aim to investigate the mechanisms of action of nano plastics (nano PS) on plants, seeds of Allium cepa were germinated for 72 h in the presence of polystyrene nano PS (50 nm size, at concentrations of 0.01, 0.1 and 1 g L−1) and, subsequently, roots were analysed by a multifaceted approach. No effect was induced by any concentration of nano PS on the percentage of seed germination while root growth was inhibited by 0.1 and 1 g L−1 nano PS. Cytological analysis of the root meristems indicated cytotoxicity (reduction of mitotic index) and genotoxicity (induction of cytogenetic anomalies and micronuclei) starting from the lowest dose. Moreover, the biochemical and histochemical analysis of oxidative stress markers gave evidence of stress induction, especially at the highest doses. Damages reported could be due to mechanical surface contact in root external layers, as evidenced by histological localization, and to the internalization of nano PS in different cellular compartments, observed under TEM. The present research underlines the hazardous nature of nano PS, that for their ability to be internalized into crop plants, can enter into different trophic levels of the food chain.

Agroecosystems represent more and more a huge long-term sink for plastic compounds which inevitably undergo fragmentation, generating micro-and nano-plastics, with potential adverse effects on soil chemistry and living organisms. The present work was focused on the short-term effects of two different concentrations of polystyrene nanoplastics (PSNPs) (0.1 or 1 g L-1 suspensions) on rice seedlings starting from seed germination, hypothesizing that possible acute effects on seedlings could depend on oxidative damage trigged by PSNPs internalization. As shown by TEM analysis, PSNPs were absorbed by roots and translocated to the shoots, affected root cell ultra-structure, the germination process, seedling growth and root mitotic activity, inducing cytogenetic aberration. Treatments were not correlated with increase in oxidative stress markers, but rather with a different pattern of their localization both in roots and in shoots, impairing H2O2 homeostasis and membrane damage, despite the adequate antioxidant response recorded. The harmful effects of PSNPs on cell biology and physiology of rice seedlings could be caused not only by a direct action by the PSNPs but also by changes in the production/ diffusion of ROS at the tissue/cellular level.

The process of soil salinization and the preponderance of saline water sources all over the world represent one of the most harmful abiotic stress to plant growth. This pointed to the importance of obtaining plants which are tolerant or resistant to salt, considering that projection of climate change for the coming years indicate an increase in temperature and rain scarcity. In the current study, the effect of NaCl was investigated on germinating seeds of Lathyrus sativus L., Vicia sativa L., Vigna radiata L. R.Wilczek and Vigna unguiculata L. Walp., by combining physiological, biochemical, biostatistical and ultrastructural analyses. Our results revealed that germination was not influenced by high NaCl concentrations, while seedling growth was affected even at low NaCl concentrations, probably due to an alteration in water uptake and in organic matter biosynthesis. Nevertheless, the synthesis of antioxidant enzymes, phenolic acids and flavonoids was registered in all species, which tended to cope with the increasing salt stress, allowing a response mechanism such as cytoplasm detoxification and cellular turgor maintenance. Besides, the ultrastructural analysis evidenced plasmolyzed cells close to cells with a normal ultrastructure with no appreciable differences among the species. This research deeply investigates the mechanism of salt-stress response focusing on species never tested before for their possible tolerance to salinity.

In the analysis by electrospray (+) of an extract of hemp sprouts put in a polypropyl-ene vial, we found a large contamination of a plastic additive. It was characterized bymultiple-stage MSnexperiments (MSMS10) and identified as tris(2,4-di-tert-butylphenyl)phosphate, also known with the synonyms F32IRS6B46, oxidizedNaugard 524, and others. The MS2MS7spectra are characterized by consecutiveeliminations of six isobutene molecules from thetert-butyl moieties, some of themalso occurring in the ion source. The first three are calculated to occur preferentiallyfrom theorthopositions, whereas eliminations from theparapositions are estimatedto be less favored at about 5-6 kcal/mol in each step. Once the first three isobutenemolecules are eliminated, the remaining three are lost from thetert-butyl moieties inparapositions (MS5MS7), yielding protonated triphenylphosphate, whose struc-ture has been confirmed by the MS2spectrum of triphenylphosphate standard: thelatter spectrum is almost superimposable with the MS8spectrum of the analyteunder investigation. MS8and MS9spectra show main losses of water and C6H4mole-cules. The MS10spectrum of precursor ions atm/z215 shows the gas-phase additionof water and methanol and ions atm/z168, attributable to the loss of a phosphorusoxide radical. Density functional theory (DFT) calculations (Becke 3LYP [B3LYP]6-311+G(2d,2p)) have been used to evaluate structure and stability of different ionicand neutral species involved in the decomposition pathways and to calculate thermo-chemical data of the decomposition reactions. This multistep mass analysis combinedwith theoretical calculations resulted to be particularly useful and effective, yieldingchemical, thermochemical, and mechanistic data of significant utility in the structuralcharacterization and identification of the unknown analyte as well as to define itsgas-phase reactivity under a multistep low-energy collision-induced dissociationregime.

In order to assess the quality and performance of bagged broccoli-raab, a recently marketed product, several nutritive parameters were determined in novel hybrid and conventional cultivars at pre- and post-packaging stages in the industrial environment. The characterization of shoots and composing organs at post-cut stage included contents of dietary fibre (DF), glycaemic carbohydrates (GC), antioxidant compounds (ACC) and capacity (AOC), which were determined by chromatographic methods and spectrophotometric assays. ACC and AOC were analysed during shelf life of bagged products. Genotype and storage effects were addressed as variability factors at fixed packaging conditions. Contents of DF and GC (39.64-34.57; 7.56-2.21 g/100 g), glucosinolates (37.47-24.63 mg/g SIN), and ACC (total phenolics: 18.64-14.92 mg GAE/g; flavonoids: 34.74-30.96 mg/g CE; flavonols: 14.62-14.08 mg QE/g), and AOC (Oxygen Radical Absorbance Capacity: 354.62-293.25 µmol/g TE; DPPHo scavenging activity: 59.35-46.14) were lower in shoots of the hybrid than marketed cultivar. In both genotypes, AOC was maximal in leaves, followed by florets and stems. The integrated analyses suggested that the hybrid genotype was better suited for fresh consumption and that increased ratio of florets/leaves vs. stem is expected to raise product antioxidant properties. The comparison of unprocessed and bagged products pointed at a value decay of most parameters except for glucosinolates and correlation analyses supported the necessity of performing multiple antioxidant assays to enhance product quality evaluation. As for shelf life, storage time was the major factor affecting antioxidant properties, while genotype and interaction effects were minimal.

In an attempt to alleviate salt-induced damage, the application of ZnO nanoparticles has been suggested. As the use of these particles has also been associated with phytotoxicity, to better clarify the effect of zinc and its possible mitigation of salt stress, we treated wheat seedlings with ZnO (nanoparticles or their bulk-scale counterparts, amended either in the growth medium, NPs and B, or sprayed on the leaves, SPNPs and SPB) with or without subsequent treatment with salt. Growth, photosynthetic parameters, zinc and ion concentration, and in situ and biochemical determination of oxidative stress in wheat leaves and/or in roots were considered. Both Zn and NaCl significantly inhibited growth and induced severe alterations in root morphology. Oxidative stress and damage decreased or increased under ZnO treatment and in saline conditions depending on the organ and on the size and mode of application of particles. In spite of the higher stress conditions often recorded in treated leaves, neither pigment concentration nor photochemical efficiency were decreased. A large variability in the effects of ZnO treatment/priming on seedling salt response was recorded, however, the presence of a cumulative negative effect of priming and salt stress sometimes observed calls for caution in the use of ZnO in protection from saline stress.

In the analysis by electrospray (+) of an extract of hemp sprouts put in a polypropylene vial, we found a large contamination of a plastic additive. It was characterized by multiple-stage MS

E171 (titanium dioxide, TiO2), an authorized foods and beverage additive, is also used in food packaging and in pharmaceutical and cosmetic preparations. E171 is considered to be an inert and non-digestible material, not storable in animal tissues, but the possible presence of TiO2 nanoparticles (NP) may present a risk to human health and the environment. We determined the presence of 15% TiO2 NP in a commercial E171 food additive product, by electron microscopy. The biological effects of E171 were assessed in Lens culinaris and Allium cepa for the following endpoints: percentage of germination, root elongation, mitotic index, presence of chromosomal abnormalities, and micronuclei. The results indicated low phytotoxicity but dose-dependent genotoxicity. We also observed internalization of TiO2 NP and ultrastructural alterations in the root systems.

The wide use of nanoparticles (NPs), gives concern about their possible negative implications in the environment and living organisms. In particular, titanium dioxide (TiO

The aim of the present work was to study, in the model system Vicia faba L., the potential stress-induced response to a commercial source of TiO2 nanoparticles (NPs)

Halloysite nanotubes (HNTs) are natural nanomaterials that are biocompatible and available in large amounts at low prices. They are emerging nanomaterials with appealing properties for applications like support for metal nanoparticles (NPs). The potential environmental impacts of NPs can be understood in terms of phytotoxicity. Current research has been focusing on HNT applications in cell or animal models, while their use in plants is limited so their ecotoxicological impact is poorly documented. To date there are no studies on the phytotoxic effects of functionalized halloysites (functionalized-HNTs). To develop a quantitative risk assessment model for predicting the potential impact of HNT-supported palladium nanoparticles (HNT-PdNPs) on plant life, an investigation was undertaken to explore their effects on seed germination, seedling development, and mitotic division in root tip cells of 2 lots of Raphanus sativus L. with different vigor. The results showed that exposure to 1500 mg/L of HNTs, functionalized-HNTs, and HNT-PdNPs had no significant influence on germination, seedling development, xylem differentiation, or mitotic index in both lots. Cytogenetic analyses revealed that treatments with functionalized-HNT significantly increased the number of aberrations in low-vigor seeds. These results suggest that low-vigor seeds represent a model for a stress test that would be useful to monitor the effects of NPs. Moreover the present study offers scientific evidence for the use of halloysite for environmental purposes, supporting the biological safety of HNT-PdNPs. Environ Toxicol Chem 2016;35:2503-2510. © 2016 SETAC.

In the analysis by electrospray (+) of an extract of hemp sprouts put in a polypropylene vial, we found a large contamination of a plastic additive. It was characterized by multiple-stage MSn experiments (MS ÷ MS10 ) and identified as tris(2,4-di-tert-butylphenyl)phosphate, also known with the synonyms F32IRS6B46, oxidized Naugard 524, and others. The MS2 ÷ MS7 spectra are characterized by consecutive eliminations of six isobutene molecules from the tert-butyl moieties, some of them also occurring in the ion source. The first three are calculated to occur preferentially from the ortho positions, whereas eliminations from the para positions are estimated to be less favored at about 5-6 kcal/mol in each step. Once the first three isobutene molecules are eliminated, the remaining three are lost from the tert-butyl moieties in para positions (MS5 ÷ MS7 ), yielding protonated triphenylphosphate, whose structure has been confirmed by the MS2 spectrum of triphenylphosphate standard: the latter spectrum is almost superimposable with the MS8 spectrum of the analyte under investigation. MS8 and MS9 spectra show main losses of water and C6 H4 molecules. The MS10 spectrum of precursor ions at m/z 215 shows the gas-phase addition of water and methanol and ions at m/z 168, attributable to the loss of a phosphorus oxide radical. Density functional theory (DFT) calculations (Becke 3LYP [B3LYP] 6-311+G(2d,2p)) have been used to evaluate structure and stability of different ionic and neutral species involved in the decomposition pathways and to calculate thermochemical data of the decomposition reactions. This multistep mass analysis combined with theoretical calculations resulted to be particularly useful and effective, yielding chemical, thermochemical, and mechanistic data of significant utility in the structural characterization and identification of the unknown analyte as well as to define its gas-phase reactivity under a multistep low-energy collision-induced dissociation regime.

A particular interest is nowadays given to natural antioxidants occurring in foods which can reduce the risk of several diseases through their protective effect. The genus Limonium is widely distributed in different salt regions of Tunisia and known in traditional medicine for the presence of highly effective viral and bacterial replication inhibitors. Limonium leaves have possible beneficial effects on human health for their antioxidant activities and free radical scavenging abilities. To exploit the potential of plants from extreme environments as new sources of natural antioxidants, we studied the extracts from leaves of eight Limonium species growing in extreme environments in Tunisia. Antioxidant molecules (polyphenols, flavonoids, flavonols, ascorbate, tocopherols), in vitro (DPPH, ORAC) and ex vivo antioxidant potential on human erythrocytes, antioxidant enzymes activities (superoxide dismutase, peroxidases, glutathione reductase) were evaluated to identify the species with the best antioxidant capacity. The results showed variability among the species considered in function of the environmental conditions of their natural biotopes, as for the antioxidants measured. In particular, L. vulgare from Oued Rane biotope, characterized by dryness and high temperatures, was the species with the highest enzymatic activity and antioxidant capacity, making it interesting as possible edible halophyte plant or as food complement.

Lentils (Lens culinaris L.) are among the most ancient seeds cultivated since prehistory. Several lentil varieties, differing for nutrients and antioxidant compounds, are cultivated in various regions of Italy. Seeds of three varieties of L. culinaris: Onano, Beluga and Villalba, differing for morphology, colour and size, were studied. Antioxidant activity and the content in total polyphenols, flavonoids and flavonols were evaluated in raw and cooked seeds and in cooking water. The same compounds were quantified in sprouts up to 4 days of germination. The highest content of polyphenols was observed in integument of Beluga seeds, flavonoids in integument of Villalba seeds, while flavonols were particularly abundant in cotyledon of Onano seeds. After seed cooking, a decrease in phytochemicals and antioxidant capacity was observed in tissues but they were partially recovered in cooking water. ·

This study focused on crop plant response to a simultaneous exposure to biosolid and TiO2 at micro- and nano-scale, being biosolid one of the major sink of TiO2 nanoparticles released into the soil environment. We settled an experimental design as much as possible realistic, at microcosm scale, using the crop Pisum sativum. This experimental design supported the hypotheses that the presence of biosolid in the farming soil might influence plant growth and metabolism and that, after TiO2 spiking, the different dimension and crystal forms of TiO2 might be otherwise bioavailable and differently interacting with the plant system. To test these hypotheses, we have considered different aspects of the response elicited by TiO2 and biosolid at cellular and organism level, focusing on the root system, with an integrative approach. In our experimental conditions, the presence of biosolid disturbed plant growth of P. sativum, causing cellular damages at root level, probably through mechanisms not only oxidative stress-dependent but also involving altered signalling processes. These disturbances could depend on non-humified compounds and/or on the presence of toxic elements and of nanoparticles in the biosolid-amended soil. The addition of TiO2 particles in the sludge-amended soil, further altered plant growth and induced oxidative and ultrastructural damages. Although non typical dose-effect response was detected, the most responsiveness treatments were found for the anatase crystal form, alone or mixed with rutile. Based on ultrastructural observations, we could hypothesise that the toxicity level of TiO2 nanoparticles may depend on the cell ability to isolate nanoparticles in subcellular compartments, avoiding their interaction with organelles and/or metabolic processes. The results of the present work suggest reflections on the promising practice of soil amendments and on the use of nanomaterials and their safety for food plants and living organisms.

The wide use of nanoparticles (NPs), gives concern about their possible negative implications in the environment and living organisms. In particular, titanium dioxide (TiO2) NPs are accumulated in biosolids (Bs) coming from wastewater treatment plants, which in turn are used as farm soil amendments and are becoming an important way of NPs entrance in the terrestrial ecosystems. In this study, to simulate a low and cumulative load of TiO2 NPs, 80 and 800 mg TiO2 per Kg of soil were spiked in the Bs prior to its addition to soil. The effects of different crystal phases of TiO2 NPs (pure anatase and pure rutile or their mixture) and their non-coated bulk counterparts (larger particles) on the availability of mineral nutrients and on the status of the bacterial communities together with the nutritional status of Pisum sativum L. plants were evaluated. Results showed the reduction, to different extents, on the availability of important soil mineral nutrients (e.g. Mn 65%, Fe 20%, P 27%, averagely), in some cases size- (e.g. P) and dose-dependent. Bacterial biodiversity was also affected by the presence of high TiO2 dose in soil. The mineral nutrition of pea plants was also altered, showing the main reduction in Mn (80% in the roots and 50% in the shoots), K, Zn, P (respectively, 80, 40, and 35% in the roots), and an increase of N in the shoots, with possible consequences on the quality of the crop. The present study gives new integrated data on the effects of TiO2 NPs in the soil-plant system, on the soil health and on the nutritional quality of crops, rising new implications for future policies and human health.

This study focused on crop plant response to a simultaneous exposure to biosolid and TiO

The yeast Pichia fermentans DISAABA 726 strain (P. fermentans) is a dimorphic yeast that under different environmental conditions may switch from a yeast-like to pseudohyphal morphology. We hypothesize that exosomes-like vesicles (EV) could mediate this rapid modification. As EV are membrane-derived vesicles carrying lipids, proteins, mRNAs and microRNAs and have been recognized as important mediators of intercellular communication. Although it has been assumed for a long time that fungi release EV, knowledge of their functions is still limited. In this work we analyze P. fermentans EV production, during growth in two different media containing urea (YCU) or methionine (YCM) where yeast-like or pseudohyphal morphology, are produced. We developed a procedure to extract EV from the neighboring biofilm, which is faster and more efficient as compared to the widely used ultracentrifugation method. Differences in morphology and RNA content of EV, suggest that they might have an active role during dimorphic transition as response to the growth conditions. Our findings are coherent with a general state of hypoxic stress of the pseudohyphal cells. This article is protected by copyright. All rights reserved.

In saline biotopes, different mechanisms may contribute to the tolerance of halophytes to high soil salinity and temperature, drought and other abiotic stresses, but their relative responses and their ecological plasticity for a given species remain unknown. In this study, we examined the responses of the salt marsh halophyte Limonium delicatulum to changing environmental conditions of its natural habitats (Sabkha “El Kalbia”, Tunisia). The specific aim of the work was to check whether statistically significant correlations could be established between particular stress response mechanisms (ion uptake, activation of antioxidant systems) and soil parameters and climatic data associated with environmental stress. The results showed that the salinity of the soil increased during the months of June, July and August (dry period), concomitantly with a strong accumulation of salt ions in the aerial parts of the plant. Moreover, the highest antioxidant capacity of this halophyte (enzymes and antioxidant molecules) was reached during the same period of the year. The remaining periods (corresponding to the rainy season) did not show a significant difference in enzymes activities and level of antioxidants, with a minimum observed in the months of January and February. These results show a remarkable effect of salinity in the natural habitat on the activity of enzymes and antioxidant molecules. Other edaphic and climatic factors could also be involved to increase antioxidant capacity, such as nutrient deficiency, temperature and precipitation.

In this study the antioxidant effect of Cannabis sativa L. seeds and sprouts (3 and 5 days of germination) was evaluated. Total polyphenols, flavonoids and flavonols content, when expressed on dry weight basis, were highest in sprouts; ORAC and DPPH (in vitro assays), CAA-RBC (cellular antioxidant activity in red blood cells) and hemolysis test (ex vivo assays) evidenced a good antioxidant activity higher in sprouts than in seeds. Untargeted analysis by high resolution mass spectrometry in negative ion mode allowed the identification of main polyphenols (caffeoyltyramine, cannabisin A, B, C) in seeds and of ?-6 (linoleic acid) in sprouts. Antimutagenic effect of seeds and sprouts extracts evidenced a significant decrease of mutagenesis induced by hydrogen peroxide in Saccharomyces cerevisiae D7 strain. In conclusion our results show that C. sativa seeds and sprouts exert beneficial effects on yeast and human cells and should be further investigated as a potential functional food.

We report the spectrophotometric determination of total polyphenols, flavonoids, glucosinolates and antioxidant activity in seeds, seedlings and leaves of Tuscan black kale. The highest content of phytochemicals was observed in 10 days sprouts and antioxidant activity was maximum in 2, 4 days seedlings. Identification and characterisation of phytochemicals were performed by mass spectrometry (MS), high resolution and tandem MS with electrospray ionisation mode. Low-molecular-weight metabolites were evidenced in seeds while metabolites at high m/z range were detected in cotyledons and leaves. MS spectra evidenced different phenolic compounds (flavonoid caffeoyl glucose, hydroxycinnamic acid sinapine) and glucosinolates (glucoerucin, glucobrassicin and glucoraphanin) in function of developmental stage; galactolipids ω3 and ω6 were observed in leaves. Identification of stages with the highest phytochemicals content encourages the consumption of black kale sprouts and young leaves. Our research can support food and pharmaceutical industries for production of health promoting products from black kale.

The effects of different copper concentrations on percentage germination, increase in fresh weight and radicle growth of Vicia sativa L. seeds were studied. Physiological studies showed that the germination rate was not affected up to a concentration of 5 × 10−3 M, but already at 10−3 M the copper stopped root elongation. Structural and ultrastructural observations of embryo and cotyledon reserve mobilization showed that inhibition of radicle growth at 10−3 M Cu concentration cannot be ascribed to nutrient shortage but probably to an effect of copper on radicle cell division and elongation. In seeds treated with 5 × 10−3 M CuBr2, the copper completely inhibited cotyledon protein mobilization, so that embryo protein mobilization supported normal growth of the radicle up to 30 h after imbibition. The particular protein content of adjacent cotyledon cells is also discussed.

Seeds of Mucuna pruriens (L.) DC. (Fabaceae) were analyzed for protein composition of protein bodies isolated from cotyledons. Protein bodies were successfully separated by Lympholyte and those of dry seeds, observed by scanning electron microscope, were elliptical or spherical in shape with a diameter of 5-12 μm. Protein content in dry seed protein bodies was 10.6 mg/g dry weight. Globulin was the largest protein fraction isolated (62.5 %), followed by albumin (18.3 %), glutelin (15.8 %) and prolamin (3.4 %). The prolamin fraction and high glutelin content are uncommon in legumes. SDS-PAGE of albumins, globulins, prolamins and glutelins provided different band numbers and molecular weights under reducing and non reducing conditions and suggested that the albumin fraction is rich in disulphide bonds.

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 c-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, a-TOC and total fatty acid content. An increase in ROS release from seed coats and in c-TOC, d-TOC, c-TOC-3 content in embryonic axis was also observed. The use of c-TOC and total fatty acids in embryonic axis as parameters of seed quality evaluation during storage was suggested. Keywords Ageing Fatty acids Phytosterols Raphanus sativus ROS release Vitamin E

Under stress integrated germination test (SIGT), seeds undergo osmo-saline stresses, which enable to detect differences in vigour of long-term stored seeds with high germination percentage (G%). The quality of Brassica villosa subsp. drepanensis seeds stored in a genebank (at -20°C for 16 years) was compared with seeds at harvest by standard germination tests (GT), SIGT and cytogenetic analysis. No differences were detected in G% and mean germination time under GT. Conversely, SIGT performed with NaCl -0.9MPa osmotic potential did not influence G% at harvest but reduced that of stored seeds, SIGT at -1.4MPa reduced G% of both. Cytogenetic analysis showed reduction of mitotic index, appearance of chromosomal aberrations and smaller nucleoli in stored seeds compared with harvest seeds germinated in water. SIGT at -0.9MPa had no effect on mitotic index, but increased chromosome aberrations and nucleoli number. SIGT at -1.4MPa inhibited G% of harvest and stored seeds, reduced mitoses in harvest and completely prevented it in stored seeds. The results indicate that GT does not faithfully reflect the quality of stored seeds, with misinterpretation of their vigour, whereas SIGT and cytogenetical parameters are sensitive, reliable and inexpensive methods for early prediction of genetic erosion in germplasm banks.

Mature pollen grains of Sambucus nigra L. are tricolporate, isopolar, 12.5 μm wide, 25 μm long, and tricellular. They have a tectate exine, a bilayered nexine, and a thin intine. The vegetative cell has abundant rough endoplasmic reticulum, many well‐structured mitochondria, numerous apparently inactive dictyosomes, immature dividing, and starch‐filled plastids and lipid bodies. The sperm cells have few and poorly structured organelles. They are linked by cytoplasmic bridges, indicating a persistent physiological unit, and are surrounded by fibrillar polysaccharide material. Freshly released pollen is 95% viable. Three‐ and 4‐year‐old pollen grains stored at −20°C are 78.2% and 43% viable, respectively. In vitro germination requirements and pollen tube growth are more similar to those of bicellular pollen than to those of tricellular pollen.

The present research reports a biochemical and micro-submicroscopic analysis of copper effect on reserve mobilization during germination of Phaseolus vulgaris L. var. soisson nain hatif seeds. Dry embryonic cells are rich in protein bodies and little starch grains. In Cu-treated embryos copper inhibited 50 % of albumin and globulin mobilization after 72 h imbibition. The severe alterations in treated embryo cells, observed by electron microscope, were probably the cause of the inability to utilize the amino acids freed by protein mobilization and so possibly the cause of the inhibition of P. vulgaris embryonic axis elongation.

Copper bromide (CuBr2) effects on seed germination and plantlet development of Vicia sativa L. are evaluated through mitotic index, chromosome aberrations, nucleolar activity and mineral profile. CuBr2 induces a significant presence of micronuclei, sticky and c-metaphases, anaphase bridges and chromosome breaks. Increased number of nucleoli and scattering of AgNOR proteins from the nucleolus in the nuclear surface at CuBr2 1 mM and in the cytoplasm at CuBr2 5 mM, goes along with the decrease of root growth. In V. sativa embryo the content of many macro and micronutrients increases up to copper 1 mM in agreement with reserve mobilization while at CuBr2 5 mM some elements are present in lower amount. We hypothesize that inhibitory effects observed at 5 mM are due either to a nutrient shortage or to a direct influence of copper on root cell division, evidenced by low mitotic index, high occurrence of chromosome aberrations and loss of material from the nucleolus.

Summary Dehiscing pollen grains of 901 species belonging to 104 dicot families and 15 monocot families were scored for starch reserves. Starch grains showed different physico-chemical properties i.e. colour after iodine - potassium iodide staining and birefringence or otherwise under polarized light. Further tests performed in a limited number of species revealed other kinds of carbohydrate reserves in the cytoplasm but outside plastids. From these observations, it results that carbohydrate reserves may be stored in plastids only (amyloplasts), in the cytoplasm but not in plastids, or in both. These kinds of pollen reserves are only partly in line with systematics, as only certain families consistently show the same type of reserve. These and previous findings suggest that the presence of polysaccharides in the cytoplasm prevents rapid decrease in viability due to desiccation. In this sense, our findings are in line with ecophysiological adaptations such as the respective pollination syndrome.

Summary Radish etiolated cotyledons were excised from the seed after 24 hours of imbibition and incubated for different times on filter paper satured with NaCl solutions or water. After the incubation of different times in NaCl, the cotyledons were thoroughly rinsed in water and grown in darkness on filter paper satured with distilled water. Continuous presence of NaCl is not a prerequisite for optimal growth. Several effects of the NaCl solutions are less intensive if the cotyledons are treated with NaCl for a relatively short time and then grown in water.

The purpose of this study was to determine tocopherol (Toc), fatty acid and phytosterol content in dry seeds of nine wild taxa of Sicilian Brassica sect. Brassica. Analyses were performed by gas chromatography (GC)-mass spectroscopy (MS). The biochemical analysis of dry seeds showed that in the nine taxa examined, α - and γ -Toc were the major isomers, δ - Toc was present in traces and β -Toc was never found. All taxa had a similar fatty acid composition with C 16:0 (357.2 mg kg − 1 DW), C 18:1 (393.94 mg kg − 1 DW) and C 22:1 (618.29 mg kg − 1 DW) acids present in higher amount. Unsaturated fatty acids (52.5–66.5%) were more abundant than saturated (33.5–47.5%) ones relative to the total fatty acids. Moreover, sitosterol was the most abundant (61.7–69.6%) of all phytosterols in all species and subspecies, followed by brassicasterol and campesterol at 16.0–19.3% and 11.9–20.7%, respectively. Total Toc content and the relative percentage of single isoforms were useful to discriminate seeds of B. incana , B. macrocarpa , B. rupestris group and B. villosa group. On the basis of these results, B. villosa subsp. villosa , B. villosa subsp. bivoniana , B. villosa subsp. drepanensis and B. villosa subsp. tinei can be ascribed to the B. villosa group. These taxa, having the highest T-Toc, γ -Toc and phytosterol contents, are potentially the most bioactive ones, and could, therefore, be exploited in breeding programmes to develop genotypes with putative positive effects on human health.

The mobilization of storage reserves, with particular emphasis on storage proteins of Mucuna pruriens (L.) DC., cotyledons, and embryo was investigated from the ultrastructural and biochemical points of view. Proteins and starch were the two main storage substances in cotyledons, and proteins and lipids were the main ones in the embryo. Embryo protein bodies were smaller and fewer in number than those of cotyledons. Structural and ultrastructural data determined between 24 and 48 h after imbibition and between 48 and 72 h after imbibition, the end of significant embryo and cotyledon protein mobilization, respectively, indicating more precocious storage protein mobilization in the axis than cotyledons. Moreover, storage protein mobilization in embryo and cotyledons occurred before the end of germination. Water soluble proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, producing 29 bands with molecular weights from 14 to 90 KDa. Embryo extract contained more proteins than cotyledon extract, contained seven characteristic bands, and showed a higher variability of the optical density trend than cotyledon.

The biological effects of an anionic synthetic surfactant (ABS) and a non-ionic surfactant (Citowett) were evaluated using roots of onion bulbs ( Allium cepa L.). Three parameters were studied: (1) root elongation, (2) the mitotic index, and (3) chromosome aberrations. The effects of increasing concentrations of surfactants were: (1) reduction in root length; (2) decrease in mitotic index and (3) induction of a high level of chromosome aberrations. The effects of ABS and Citowett suggest caution with regard to inappropriate use of these substances.

Peroxidase activity was assayed in crude extracts of integument, cotyledons and embryo axis of radish seeds, deteriorated under accelerated ageing conditions. Over five days of ageing, in which germination decreased from 100 to 52%, the enzyme activity in integument was higher than that in other seed parts, increasing in the first days of ageing and then decreasing sharply in extremely aged seeds. Polyacrylamide gel electrophoresis analysis showed four peroxidase isoenzymes with MM of 98, 52.5, 32.8 and 29.5 kDa in the embryo axis of unaged seeds, and only the 32.8 and 29.5 kDa MM isoforms in the integument and cotyledons. In these parts of the seed, only the 29.5 kDa MM isoenzyme increased in activity in early days of ageing and decreased thereafter. In the embryo axis, the 29.5 kDa MM isoenzyme activity increased slowly in the first day of ageing, while the 98 and 52.5 kDa MM isoenzyme activities disappeared. A cytochemical localization of peroxidase activity in the various tissues showed that main differences between unaged and extremely aged seeds occurred in the embryo axis.

Peroxidase (POD, EC 1.11.1.7) activity, cellular localization and isozyme patterns were investigated in the seed integument, cotyledon and embryo axis of Brassica oleracea cv. Cappuccio during pregermination and seedling growth. Seeds started to germinate after 24 h of imbibition. POD activity was localized in the pigmented layer of the integument and in procambial strands of the cotyledon and embryo axis in the first 24 h of imbibition. It was localized in the integumental cells of palisade, pigmented and aleurone layers and in epidermal, meristematic, procambial cells and xylem elements of the root and hypocotyl after 48 h of imbibition. POD activity increased during germination and early seedling growth: in the integument, it reached a maximum value after 72 h of imbibition, in the embryo axis and cotyledons, it increased up to 144 h of imbibition. The increase in peroxidase activity was accompanied by the appearance of new isozymes correlated with the development of seedling tissues. The isozyme profile was characterized by nine peroxidases: isoperoxidase of 50 kDa peculiar to integuments, that of 150 kDa to cotyledons and that of 82 kDa to the embryo axis. During pregerminative phase isozymes of 84 kDa were detected in the integument and cotyledons, of 48.5 kDa in the embryo axis. After germination, peroxidase activity and the complexity of the isozyme pattern increased, suggesting that they play a relevant role after rupture of the integument.

Viability, germination and tube length were investigated in pollen grains of field-grown 'Summerred' apple trees (Malus domestica Borkh) exposed to deionized water, rainfall or simulated acid rain at pH 5.6, 4.0 and 3.0. Pollen viability and germination significantly decreased with lower values of pH and with increasing number of treatments. The effects of pH 5.6 and natural rainfall were not significant. Electron microscope investigation of vegetative pollen cells of plants exposed to acid rain at pH 4.0 and 3.0 showed modified features in mitochondria, plastids and endoplasmic reticulum.

(1996). Spermatogenesis in Asplenium trichomanes ssp. trichomanes. Giornale botanico italiano: Vol. 130, No. 4-6, pp. 945-947.

The basic anatomy of lateral twig insertion onto the main branch in both healthy and damaged Quercus cerris L. trees was studied. An abscission zone is always present: in healthy trees it is formed by a smaller number of cell layers than in damaged ones, where it is more evident with many layers of cells. Cells of the abscission zone are roundish, with many intercellular spaces between them; cell walls are thin, non-lignified and without secondary walls. No starch was found in cells of the abscission zone, where, instead, a few scattered calcium oxalate druses are seen.

Pollen grains collected from trees of Quercus ilex L. belonging to different classes of new type damage were analyzed. Five parameters were taken into account: viability, germinability, tube lengthening, grain volume of the dehydrated state and after rehydration. Damage to mother plants clearly influences the above mentioned parameters, causing a reduction in pollen grain viability, germinability, and in the ability of the grains to rehydrate.

Pollen germination and tube growth are among the most sensitive responses to atmospheric pollution. Both these are inhibited by the acidity of the growth medium. Pollen grains from two species ( Pinus cembra L. and Sambucus nigra L.) were germinated in media over a range of pHs (5·0, 4·5, 4·0, 3·5, 3·0 and 2·5) and six types of acidity (H 2 SO 4 , HNO 3 , H 2 SO 4 : HNO 3 in 1:1, 2:1, 3:1 and 5:1 ratio). Pollen of the Gymnosperm is shown to be more resistant to acidity in the medium. Sulphuric acid alone and the ratio 2:1 with nitric acid are demonstrated to be the more harmful for P. cembra and S. nigra , respectively. The latter species was sensitive to all mixtures, particularly in respect to germination percentage.

A simple method to enhance the staining of cell wall components for fluorescence microscopy is described. In stems of Nicotiana tabacum and needles of Pinus eldarica lignin, the cuticle and unsaturated lipids are indicated by a purple-red fluorescence while pectocellulosic components fluoresce pale blue.