Improved protocol for Agrobacterium mediated transformation of tomato and production of transgenic plants containing carotenoid biosynthetic gene CsZCD

Improved protocol for Agrobacterium mediated transformation of tomato (Lycopersicon esculentum) Micro-Tom was developed to use in corporation of the carotenoid biosynthetic genes CsZCD (Crocus zeaxanthin 7,8-cleavage dioxygenase). From these experiment, a transformation methodology using explants from cotyledons cultured for 1 day on the medium with zeatin 2 mg/L, IAA 0.1 mg/L, carefully submerged in the Agrobacterium inoculum for 20 min, then concultured with the agrobacterium for 3 days on the same medium, followed by a transfer to the same medium with 500 mg/L cefotaxin for 3 days and then by a transfer to the same medium with 100 mg/L kanamycin and 500 mg/L carabenillin for 6–8 weeks and resulted in a greater than 20% transformation efficiency in the concentration of Agrobacterium OD600 = 0.2 tested. In this transformation method, no feeder layers were used and the subculture media was minimal. Among the Agrobacterium concentrations of OD600 = 0.2, 0.5 and 1.0, the best transformation efficiency, 20.87%, was obtained by using OD600 = 0.2, which was significantly higher than that of OD600 = 1.0. The presence of the inserted target genes was checked using a rapid and efficient PCR test. The protocol was successfully employed in the production of transgenic Micro-Tom tomato containing the carotenoid biosynthesis CsZCD under constructive promoter. This procedure represents a simple, efficient and general means of transforming tomato.     

Advanced oxidation protein products are generated by bovine neutrophils and inhibit free radical production in vitro

Despite the recognised importance of oxidative stress in the health and immune function of dairy cows, protein oxidation markers have been poorly studied in this species. The current study aimed to characterise markers of protein oxidation generated by activated bovine neutrophils and investigate the biological effects of advanced oxidation protein products (AOPP) on bovine neutrophils. Markers of protein oxidation (AOPP, dityrosines and carbonyls) were measured in culture medium containing bovine serum albumin (BSA) exposed to neutrophils. The effect of AOPP-BSA on generation of reactive oxygen species (ROS) was assessed by chemiluminescence. Activation of caspases-3, -8 and -9 and the presence of DNA laddering were used as apoptosis markers.Greater amounts of AOPP were generated by phorbol myristate acetate (PMA)-activated than non-activated neutrophils (1.46 ± 0.13 vs. 0.75 ± 0.13 nmol/mg protein, respectively; P < 0.05). Activated neutrophils and hypochlorous acid generated slightly different patterns of oxidized protein markers. Exposure to AOPP-BSA did not stimulate ROS production. Activated neutrophils generated a lesser amount of ROS when incubated with AOPP-BSA (P < 0.001). Activation with PMA induced a loss of viable neutrophils after 3 h, which was greater with AOPP-BSA incubation (P < 0.05). Detectable amounts of active caspases-3, -8 and -9 were found in nearly all samples but differences in caspase activation or DNA laddering were not observed comparing treatment groups. Apoptosis was unlikely to be responsible for the greater loss of PMA-activated neutrophils cultured in AOPP-BSA and it is possible that primary necrosis occurred. The results suggest that accumulation of oxidized proteins at an inflammatory site might result in a progressive reduction of neutrophil viability.     

Study of stomatal parameters for selection of drought resistant varieties in Triticum durum DESF

An analysis was made of the stomatal apparatus of the leaves of five Italian durum wheat varieties, grown under different natural rainfall regimes.Rainfall had a significant influence on both development of the leaves and frequency and size of the stomata. Results were examined with respect to yield stability. Correlations between examined traits were calculated.     

Estimation of color of durum wheat. Comparison of WSB, HPLC, and reflectance colorimeter measurements

Color is an important parameter involved in the definition of semolina and pasta quality. This character is mainly due to natural pigments (carotenoids) that are present at different levels in cereals and cereal products, due to botanical origin, growing conditions, distribution in the kernel, and technological processes. In food industries, color measurements are usually performed by means of automatic instruments that are rapid and safe, as alternatives to the chemical extraction methods. In this study, automatic measurements (CIE, color-space system L, a, b), water-saturated butanol (WSB), and HPLC determinations have been applied to evaluate the carotenoid content in whole meals and respective semolina samples produced from wheat cultivated in the years 2001 and 2002. In whole meals, total carotenoids, determined by HPLC, were about 3.0 microg/g (2001) and 3.5 microg/g (2002) calculated on dry weight (dw) and about 3.0 and 3.2 microg/g dw in corresponding semolina samples. The b values for the same period were 19.78 and 15.75, respectively, in raw materials and 20.03-21.67 in semolina. Results have confirmed lutein and beta-carotene as the main components mainly responsible for the yellow color in wheat grains. The ability of the index b to express natural dyeing was dependent on sample characteristics as demonstrated by the relationships found between this index and pigments, although the best correlation resulted between HPLC and WSB.     

Relationship between oxidative stress and the success of artificial insemination in dairy cows in a pasture-based system

This study was designed to evaluate whether the outcome of artificial insemination (AI) was affected by the metabolic and oxidative status of dairy cows. Seventy-nine inseminations in 40 cows, were classified, on the basis of blood progesterone (P4) and pregnancy-associated glycoprotein (PAG) concentrations and clinical confirmation of pregnancy into, three categories: (1) positive (AI+, resulted in pregnancy, n=26; 33%), (2) negative (AI-, did not result in pregnancy, n=49; 62%), and (3) embryonic mortality (EM, n=4; 5%). Reactive oxygen metabolites, biological antioxidant potential, oxidative stress index, body condition score, glucose, total proteins, albumin, urea, non-esterified fatty acids (NEFAs), cholesterol, triglycerides, haptoglobin and advanced oxidative protein products (AOPPs) were measured on the day of AI (day 0), and 30 and 42days later. Cows with EM had lower BCS scores (2.5) than AI+ (2.8) and AI- (2.9) cows (P<0.05). During the post-partum period, body condition score (BCS) increased and NEFAs decreased (P<0.05) suggesting a recovery from the negative energy balance (NEB). The only significant differences found were that the mean concentration of AOPPs was higher and that of albumin lower in EM cows than in AI+ and AI- (P<0.05) animals. Plasma concentration of reactive oxygen metabolites and biological antioxidant potential were not related to AI outcome. Further studies are required to confirm this finding and to clarify the role of oxidative status on cows' fertility.     

Bioactive Compounds in Wild Asteraceae Edible Plants Consumed in the Mediterranean Diet

Plant Foods for Human Nutrition - Three wild edible plant species belonging to the Asteraceae family, Crepis vesicaria L. (s.l.), Sonchus asper (L.) Hill s.l., and Sonchus oleraceus L., usually...     

Dissection of antioxidant activity of durum wheat (Triticum durum Desf.) grains as evaluated by the new LOX/RNO method

Antioxidant activity (AA) of durum wheat (Triticum durum Desf.) grains was studied using the innovative LOX/RNO method, able to simultaneously detect different antioxidant mechanisms, and the TEAC assay, one of the most widely used assays. Insoluble-bound and free-soluble phenols, hydrophilic and lipophilic compounds were extracted from eight different whole flour samples; extracts were analyzed for AA and their content in several antioxidants. The LOX/RNO method measured very high AA values, with the highest ones [850–1500 μmol Trolox eq./g whole flour (dry weight)] for insoluble-bound phenolic extracts, highly correlated to total phenolic (r = 0.761, P < 0.001) and ferulic acid (r = 0.816, P < 0.001) contents. Hydrophilic and lipophilic extracts showed lower AA [70-140 and 40–60 μmol Trolox eq./g (dry weight), respectively], highly correlated to flavonoid (r = 0.583, P < 0.01) and protein (r = 0.602, P < 0.01), as well as β-tocotrienol (r = 0.684, P < 0.05) contents, respectively. Interestingly, the LOX/RNO method suggests that insoluble-bound phenolic compounds may exert very strong synergistic interactions within the extract. Contrarily, the TEAC assay did not correlate to any antioxidant content, resulted unable to highlight differences among samples, measured much lower AA values and did not suggest synergism. The use of the LOX/RNO method is useful to unearth new properties of phytochemicals from durum wheat grains, potentially giving health benefits.     

Isolation and characterization of Avenin-like protein type-B from durum wheat

Proteomic techniques were applied for the investigation of “avenin-like protein type-B” in tetraploid wheat. Protein was extracted from endosperm, according to a classical chromatographic procedure and its identity confirmed by liquid chromatography-tandem mass spectrometry analysis. The characterization of avenin-like protein type-B was in good agreement with the gene-derived sequence, with the exception of glutamine as N-terminus. The presence in the glutenin extract and the high number of cysteine residues suggested that avenin-like protein type-B is integrated into gluten polymers via inter-chain disulphide bonds. This raises the question of whether the protein could play a role in determining the functional properties of gluten.