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A three-day pretreatment of olive somatic embryos (SE) with 0.75 M sucrose, combined with cryoprotection (0.5M DMSO, 1M sucrose, 0.5M glycerol and 0.009 M proline) and controlled rate cooling, supported regrowth (as 34.6% fresh weight gain) and resumption of embryo development after cryopreservation. Pretreatment with mannitol or sorbitol did not support regrowth. Profiles of sugars, proline, antioxidant enzymes, Reactive oxygen species (ROS), secondary oxidation products and ethylene were constructed for the most successful (0.75 M) pretreatment series. Sucrose was the optimal pretreatment for supporting recovery, it also elevated glutathione reductase (GR) activity compared to controls, whereas superoxide dismutase (SOD), catalase and guaiacol peroxidase activities remained relatively unchanged. Superoxide dismutase activity was higher in SE pretreated with sucrose, compared with those pretreated with polyols; H(2)O(2) was enhanced in SE pretreated with sorbitol and sucrose compared to mannitol. The overall trend for ethylene and OH production revealed their levels were highest in SE pretreated with polyols albeit, for individual treatments this was not always the case. Generally, pretreatments did not significantly change embryo secondary oxidation profiles of ThioBarbituric Acid Reactive Substances (TBARS) and Schiff's bases. In combination these studies suggest oxidative processes may influence regrowth of cryopreserved olive SE and that optimal pretreatments could, in part, increase tolerance by an overall enhancement of endogenous antioxidants (particularly GR), proline and sugars., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Summary: Hypocotyl segments of 7-day old seedlings of flax (Linum usitatissimum L.) cultivars Atalante, Flanders, Jitka, Szegedi 30 and Super were screened for organogenesis (shoot and root induction) and embryo-like structure production. A non-destructive assay for hydroxyl radicals (ising DMSO as a radical trap, was used to determine ion during tissue culture and morphogenesis. Desferrioxamine, an inhibitor of Fenton reaction, and 4-hydroxy-2-nonenal, a cytotoxic lipid peroxidation product, were exogenously applied to flax cultures to determine the effect of antioxidative and prooxidative status on morphogenetic responses induced through the exogenous application of plant growth regulators. Flax genotypes varied in their response to treatments after exposure to different plant hormones. Hydroxyl radical (tion correlated with morphogenetic responses and this was affected by plant hormones. Desferrioxamine and 4-hydroxy-2-nonenal also moderated morphogenetic responses and influenced hydroxyl radical formation during in vitro propagation. [Copyright &y& Elsevier]

Abstract: Cold tolerance studies are required to improve understanding of how plant tissues survive and regenerate from cryogenic temperatures. Ribes genotypes with different survival responses following cryopreservation were examined to determine the role of oxidative stress and ethylene in cryo-injury. In vitro shoot cultures of Ribes ciliatum (cryo-sensitive) and Ribes nigrum (cryo-tolerant) were analysed for antioxidant status, hydroxyl radicals and ethylene production at different stages of an encapsulation-dehydration cryopreservation protocol. Differential genotypic responses occurred during sucrose-simulated cold acclimation, where tolerance was associated with greater increases in hydroxyl radical activity, antioxidant status, phenolic accumulation, anthocyanin pigmentation, and protein SH group status. Elevated antioxidant levels persisted through recovery in the more tolerant genotype, while no changes in oxidative stress markers were found in shoots recovered from the sensitive genotype. Genotypic differences in the production of the stress hormone ethylene also occurred during recovery, where the cryo-sensitive genotype produced more ethylene than the tolerant species. This study indicates elevated antioxidant status and phenolic accumulation may be determinants of cryogenic stress tolerance and that their manipulation could improve recovery after cryopreservation. [Copyright &y& Elsevier]

Abstract: The 1-methyl-2-phenylindole colorimetric assay is considered specific for malondialdehyde (MDA) and 4-hydroxynonenal (HNE) in mammalian systems, but its specificity in plant tissues is unknown. This study demonstrates that the assay produces a purple/blue chromophore with an absorbance peak at 586nm for a malondialdehyde standard, while aqueous extractions from Ribes spp. Beta vulgaris, and Lycopersicon esculentum tissues produce an orange chromophore with an absorbance maximum at 450nm and a large shoulder that extends to 700nm. No distinctive MDA peak was discernable in plant samples at λ=586nm and absorbance was attributed to background interference. The reaction between sucrose and 1-methyl-2-phenylindole produced an orange chromophore with a spectrum similar to those obtained from plant extractions, suggesting that simple sugars are the likely source of background interference. This study demonstrates that the 1-methyl-2-phenylindole colorimetric assay is non-specific for detecting MDA and HNE in plants and its use is cautioned due to interference, particularly from sugars. [Copyright &y& Elsevier]

Abstract: A simple and reproducible algal tissue extraction protocol and a total antioxidant, radical cation decolourisation spectroscopic assay has been developed. This was applied to ascertain the potential for using algae and their stress responses as biological and biochemical indicators of environmental impacts in urban ponds. Total antioxidant and soluble protein profiles were constructed for the green alga Cladophora glomerata, which was seasonally sampled, from three sustainable urban drainage systems (SUDS) ponds located in an industrial and residential development created on a former Greenfield area in Fife, Scotland. Algal antioxidant profiles were impacted by a combination of lifecycle and pond structural parameters, including: seasonal sampling time, pond site and pond structure (e.g. inlet, outlet). The assay, as applied to algae as primary producer–indicator organisms is recommended for use in aquatic environmental monitoring programmes, which have a requirement to maintain the health status of disturbed aquatic urban ecosystems. [Copyright &y& Elsevier]

Abstract: A robust spectroscopic method for determining total antioxidant activity in aqueous extractions has been applied to tissues from diverse woody plant species, including seeds of Coffea arabica and in vitro shoots from Ribes nigrum, Picea sitchensis and Shorea leprosula. The assay involves scavenging of an ABTS [2,2′-azinobis-(3-ethyl-benzothiazoline-6-sulphonic acid)] radical generated by the reaction of potassium persulphate with ABTS to produce an ABTS●+ chromophore (λ=734 nm). Antioxidants reduce ABTS●+ back to ABTS with a concomitant decrease in absorbance. Aqueous extractions from C. arabica and S. leprosula had considerably higher (110–205 μmol Trolox eq. g–1 FW) total antioxidant activities than P. sitchensis and R. nigrum (6–11 μmol Trolox eq. g–1 FW). Further studies in two of these species showed that the inclusion of water-insoluble polyvinylpyrrolidone during aqueous tissue extraction enabled the combined phenolic and alkaloid antioxidant activity to be determined. These fractions accounted for 85% and 60% of total antioxidant activity for C. arabica seeds and R. nigrum shoots, respectively. The ABTS radical scavenging assay is presented herein as a robust method for determining total antioxidant activity in germplasm from diverse woody plant tissues and species. Its applicability to study oxidative stress in tissue cultures and germplasm employed in plant biotechnology, breeding and stress physiology programmes is discussed. [Copyright &y& Elsevier]