Ana Cunha

Somatic embryogenesis, organogenesis and callus growth kinetics of flax

The effects of plant growth regulators (PGR) on calli induction, morphogenesis and somatic embryogenesis of flax were studied. The organogenic and callus formation capacity were assessed for different types of source explants. Root and shoot explants were equally good material for calli production but the former produced calli without shoot regeneration capacity. Under the experimental conditions tested, 2,4-dichlorophenoxyacetic acid (2,4-D) + zeatin was the most efficient PGR combination on calli induction and biomass production. The calli were green but with no rhizogenic capacity. In contrast, and at similar concentrations, indole-3-butyric acid (IBA) + kinetin induced white or pale green friable calli with a good root regeneration capacity (60%). A factorial experiment with different combinations of 2,4-D + zeatin + gibberellic acid (GA3) levels revealed that the direction of explant differentiation was determined by specific PGR interactions and concentrations. The results from these experiments revealed that the morphogenetic pathway (shoot versus root differentiation) can be manipulated on flax explants by raising the 2,4-D level from 0.05 to 3.2 mg l−1 in the induction medium. The induction and development of somatic embryos from flax explants was possible in a range of 2,4-D + zeatin concentrations surrounding 0.4 mg l−1 2,4-D and 1.6 mg l−1 zeatin, the most efficient growth regulator combination.

Photosynthesis by six Portuguese maize cultivars during drought stress and recovery

Photosynthesis, chlorophyll fluorescence, and leaf water parameters were measured in six Portuguese maize (Zea mays L.) cultivars during and following a period of drought stress. The leaf relative water content (RWC) responded differently among cultivars but except for cultivar PB369, recovered close to initial values after watering was restored. Photosynthetic rate and stomatal conductance decreased with drought but more slowly in cultivars PB269 and PB260 than in cultivars AD3R, PB64, PB304 and PB369. Water use efficiency (WUE) decreased during the water stress treatment although with cultivar PB260 the decrease was marked only when the RWC fell below 40%. Recovery of WUE was seen with all cultivars except PB369. The maximum quantum efficiency of photosystem II, the photochemical quenching coefficient, the electron transport rate in PSII and the estimated functional plastoquinone pool tended to decrease with drought, while the non-photochemical quenching coefficient increased. The parameters estimated from chlorophyll fluorescence did not recover in PB369, during re-watering. The results show that PB260 and PB269 were the most tolerant and PB369 was the least tolerant cultivars to water stress. The variation found among the cultivars tested suggests the existence of valuable genetic resources for crop improvement in relation to drought tolerance.

Responses of primary production, leaf litter decomposition and associated communities to stream eutrophication

We assessed the eutrophication effects on leaf litter decomposition and primary production, and on periphytic algae, fungi and invertebrates. According to the subsidy-stress model, we expected that when algae and decomposers were nutrient limited, their activity and diversity would increase at moderate levels of nutrient enrichment, but decrease at high levels of nutrients, because eutrophication would lead to the presence of other stressors and overwhelm the subsidy effect. Chestnut leaves (Castanea sativa Mill) were enclosed in mesh bags and immersed in five streams of the Ave River basin (northwest Portugal) to assess leaf decomposition and colonization by invertebrates and fungi. In parallel, polyethylene slides were attached to the mesh bags to allow colonization by algae and to assess primary production. Communities of periphytic algae and decomposers discriminated the streams according to the trophic state. Primary production decomposition and biodiversity were lower in streams at both ends of the trophic gradient.

Activities of Apiaceae Essential Oils against Armyworm, Pseudaletia unipuncta (Lepidoptera: Noctuidae)

Essential oils (EOs) from four Apiaceae species and 11 pure compounds were evaluated for their antifeedant, growth inhibitory, and insecticidal activities against Pseudaletia unipuncta (Lepidoptera: Noctuidae) fourth-instar larvae. EOs from Foeniculum vulgare subsp. vulgare var. vulgare, Anethum graveolens , Petroselinum crispum , and Cuminum cyminum were characterized by gas-chromatography (GC) and mass spectrometry. Anti-insect activity varied according to plant specie/composition, type, and exposure period. EOs from P. crispum and A. graveolens fruits, trans-anethole and cuminaldehyde, exerted acute effects on larvae feeding and growth (FDI and GI > 70%). A. graveolens , C. cyminum , and F. vulgare EOs and some of their constituents were effective by fumigation (≥ 80%). Satisfactory contact toxicities (>70%) were observed for five compounds and all EOs, except F. vulgare EOs, when tested by the filter paper impregnation method. For the most active EOs/compounds, dose-dependent toxicity was determined and inverse relationships of LC50 with time were established.

Differences in free sterols content and composition associated with somatic embryogenesis, shoot organogenesis and calli growth of flax

A single medium was used for induction of somatic embryogenesis and shoot organogenesis from flax hypocotyls. With this system, a comparative study was performed to determine the free sterols content and composition in non-organogenic, shoot organogenic and embryogenic calli as well as in regenerated shoots and somatic embryos grown under the same hormonal supplementation. The induction of somatic embryogenesis and shoot organogenesis of flax was associated with an increase of total sterols in the competent calli and an increased ratio of stigmasterol to β-sitosterol in derived embryos and shoots. On the contrary, in non-organogenic calli, the ratio stigmasterol to β-sitosterol decreased during the exponential growth phase due to a drastic rise in the β-sitosterol content. This effect as well as the calli growth were higher with 0.5 mg/l 2,4-D than with 0.6 mg/l IBA.

Ontogenic variations in free and esterified fatty acids during somatic embryogenesis of flax (Linum usitatissimum L.)

Abstract In vitro cultures of flax (Linum usitatissimum L.) were established on MS medium and four samplings were made during the 7 weeks of culture. The samples varied from the original hypocotyl segments (HS) at t0 and segments with incipient calli formation (HSC) after 2 weeks (t2), to embryogenic calli (EC), non-embryogenic calli (NEC) and somatic embryos (SE) collected after 5 (t5) and 7 weeks (t7). The respective free fatty acids (FFA) and esterified fatty acids (EFA) were extracted and analyzed, as methyl esters, by GC and GC–MS, and the data were submitted to analysis of variance (ANOVA) and PCA statistical analysis. The main FFA in all samples was 16:0, 18:0, 18:1, 18:2 and 18:3. EFA fractions were more diverse containing some less common FA. The development of SE was associated with a decrease in neutral lipids. Both the total FFA and the total EFA, as fractions of total lipids, increased with the dedifferentiation and the early calli formation leveling off thereafter (t5–t7). FA variations related to dedifferentiation of hypocotyl tissues and growth of NEC, somatic embryogenesis, and development of SE were observed. Predominant or tissue-specific FA was also found. The value of 18:3/18:2 and 18:0/18:3 ratios as potential indicators of SE autotrophy and calli embryogenic capacity is discussed.

Ontogenic variations in n-alkanes during somatic embryogenesis of flax (Linum usitatissimum L.)

Hypocotyl segments (HS) of flax seedlings germinated in vitro, were used to induce indirect somatic embryogenesis on solid medium. The composition and distribution of n-alkanes in flax tissues collected at different developmental stages were studied by capillary gas chromatography (GC) and capillary gas chromatography-mass spectrometry (GC-MS). During induction and development of callus from hypocotyl tissues a decrease in the percentage of total lipids was observed. In all types of tissue sampled - HS used as primary explants, HS with differentiating calli at the cut ends (HSC), embryogenic (EC) and non-embryogenic calli (NEC) and somatic embryos (SE) - a skewed-normal distribution of n-alkanes with a low mass range (C(13)-C(21)) were found. The highest content of n-alkanes occurred in the primary hypocotyl explants and in the early stages of callus development. Longer carbon chain n-alkanes were observed only in the mature or differentiated tissues of hypocotyls and SE. Although the n-alkane contents decreased with time, in SE and calli, a significantly lower n-alkane content was observed in EC when compared to NEC independent of the time in culture. These results suggest the utilisation of n-alkanes for heterotrophic cellular growth as well as its mobilisation from EC to developing SE.

SiO₂ nanomaterial as a tool to improve Hordeum vulgare L. tolerance to nano-NiO stress

This work was designed to assess the potential role of silicon dioxide nanomaterial (nano-SiO2) in enhancing barleys tolerance to nickel oxide nanomaterial (nano-NiO). For this purpose, plants were grown for 14days under nano-NiO (120mgkg-1) single and co-exposure with nano-SiO2 (3mgkg-1). The exposure of barley to nano-NiO caused a significant decrease in growth-related parameters and induced a negative response on the photosynthetic apparatus. However, upon nano-SiO2 co-exposure, the inhibitory effects of nano-NiO were partially reduced, with lower reductions in fresh and dry biomass, and with the recovery of the photosynthesis-related parameters. Plants growing under nano-NiO stress showed an overproduction of superoxide anion (O2.-), which favored the occurrence of oxidative stress and the enhancement of lipid peroxidation (LP), but the co-treatment with nano-SiO2 reverted this tendency, generally lowering or maintaining the levels of LP and stimulating the redox pathway of thiols. The evaluation of the antioxidant (AOX) system revealed that nano-NiO induced the accumulation of proline, along with a decrease in ascorbate in leaves. Furthermore, superoxide dismutase (SOD) activity was significantly enhanced and catalase (CAT) and ascorbate peroxidase (APX) seemed to have a pivotal role in H2O2 detoxification in leaves and roots, respectively. The response of the AOX system was even more prominent upon nano-SiO2 co-exposure, reinforcing the ameliorating functions of this nanomaterial. Overall, the present study highlighted the protective role of nano-SiO2 in barley plants under nano-NiO stress, possibly due to the Si-mediated protection against oxidative stress, by a more proactive performance of the plant AOX system.

Antioxidant and dual dose-dependent antigenotoxic and genotoxic properties of an ethanol extract of propolis

Propolis is a resinous product made by honeybees from plant-derived materials, with high content of polyphenols associated to several beneficial bioactivities with potential use as a natural food additive for preservation and as a functional food ingredient. A Portuguese propolis ethanol extract (C.EE) protected Saccharomyces cerevisiae cells from loss of viability upon exposure to H2O2, both in co- and in pre-incubation experiments. Results obtained with the comet assay suggest that lower concentrations are antigenotoxic while at higher concentrations a genotoxic effect prevails, which correlates with the cytotoxicity of high concentrations of C.EE. Flow cytometry analysis with dichlorofluorescein indicates that C.EE induced intracellular antioxidant activity in vivo. Overall the results suggest that C.EE is antigenotoxic but is also toxic at higher concentrations. This dual effect could be explained by the presence of compounds known to interfere with DNA synthesis and/or cell proliferation, such as caffeic acid phenethyl ester (CAPE) and chrysin, together with antioxidants, like kaempferol, pinobanksin and pinocembrin.

Impact of the Light Microclimate on Photosynthetic Activity of Grape Berry (Vitis vinifera): Insights for Radiation Absorption Mitigations’ Measures

IPCC’s predicted rise in mean temperatures, increase in the frequency of summer heat waves and decrease in soil water availability for the Mediterranean regions will have an impact on foliar and fruit photosynthesis. But mitigation measures aiming reducing radiation absorption by the vine canopy may pose light limitations to grape berry photosynthesis. This work focused on the influence of the light level of the canopy microenvironment where clusters develop on the photosynthetic competence of grape berry tissues (exocarp and seed integument) throughout fruit growing season by imaging PAM fluorometry. Clusters from low (LL), medium (ML) and high light (HL) microclimates were sampled from green to mature stages. Both tissues showed high maximum quantum efficiency (Fv/Fm) and photosynthetic capacity (ETRm) at the green stage, exocarp extending to mature stages while seed photosynthetic activity was more restricted to green stage. The light microclimate had a significant effect on the photosynthesis of both tissues but also in their photosynthetic phenotypes along the season. In LL, both tissues showed lower activity in all stages, higher susceptibility to photoinhibition and lack of response to short-term light acclimation; ML and HL grapes adjust their activity peaking at different light intensities, were more responsive to light changing conditions, recover better from high light. Overall, our results suggest that not only light/temperature stress conditions imposed by climate changes but also viticulture practices causing changes in canopy light microclimates may have significant impacts on grape berry photosynthesis and hence in fruit development and quality.