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ŠUMARSKI LIST 5-6/2013 str. 19     <-- 19 -->        PDF

Materials and methods
Materijali i metode
Plan of experiment – Plan eksperimenta
Greenhouse pot experiment in semi controlled conditions was conducted, of the Department of Biology and Ecology, Faculty of Science, University of Novi Sad. The experiment was a factorial design with three clones of two poplar species (Populusxeuramericana-M1 clone and Populus deltoides-B229 and PE 19/66 clones), from the Institute of Lowland Forestry and Environment (University of Novi Sad, Serbia) collection.
Heavy metals (Cu2+, Ni3+ and Cd2+) were introduced to soil and all three clones were intoxicated with various concentrations of them. Concentrations of heavy metals were determined according to the maximum permissible concentration (MPC) for given heavy metals by implemented by: i) the Regulations on permitted amounts of hazardous and harmful substances in soil and methods of their testing published in the Official Gazette of the Republic of Serbia no.23, 1994, ii) the Ordinance on the methods of organic plant production and gathering wild fruits and medicinal plants as products of organic agriculture (from: Official Gazette of RS, 23/1994, SRJ, 51/2002) iii) and given the limits for the content of certain heavy metals in soil. The values for the investigated metals concentrations were: MPC (Cu2+) =100 mg/kg, MPC (Cd2+) =3 mg/kg, MPC (Ni3+) =50 mg/kg, respectively.
Sandy fluvisol soil in pots was contaminated with the heavy metal treatments presented in table 1. Each treatment was set up in three replicates. The land was treated and left for three months to form inner microbiological environment. Metals were added as a nitrate salt, Cu(NO3)2, Cd(NO3)2 and Ni(NO)3, so they made the exact concentration calculated per 100 kg soil and were dissolved in deionized water and sprayed onto the soil, which was thoroughly homogenized by mixing and placed in pots of 10 kg. In each pot, four poplar plants were planted as two-year-old seedlings, so with a single treatment (one concentration of a metal) 12 plants (four plants of each clone) were contaminated. Also, control poplars plants of each clone were planted in soil not being treated with heavy metals. Shoots of poplar clones were sampled and a series of extracts was prepared for in vitro analysis.
Preparation of plant extracts – Priprema biljnih ekstrakata
Plant extracts were made from 2 g of plant material (shoots) homogenized with quartz sand and suspended in 10 cm3 0.1 mol/dm3 K2HPO4 at pH 7.0 placed into a cold porcelain mortar and macerated for 2–3 minutes.
Homogenate was centrifuged for 10 min at 4000 g (Quy Hai et al., 1975). The resulting supernatant was used for different antioxidant and scavenger determinations: SOD activity and soluble protein content, total antioxidant activity FRAP method and lipid peroxidation through the determination of malonyldialdehyde MDA.
In vitro studies of extracts poplar clones – In vitro analize ekstrakata klonova topola
Total antioxidant capacity of shoots extracts was estimated according to the FRAP (Ferric Reducing Antioxidant Power) assay (Benzie and Strain, 1999). Total reducing power is expressed as FRAP units. FRAP unit is equal with 100 μmol/dm3 Fe2+. FRAP value was calculated using the formula:
                FRAP value=ΔAsample/ΔAstandard
Lipid peroxidation (LP) was determined by measuring amounts of malonyldialdehyde MDA which is one of its end-products and which is quantified by thiobarbituric acid (hereinafter: TBA) method (Placer et al., 1966). The values were given as nmol of MDA per mg of soluble proteins.
Soluble protein content was determined by the Bradford method (1976) and expressed as mg protein per g of dry weight. Absorbance reading was performed at 595 nm.
The total activity of superoxide dismutase was assayed by monitoring the inhibition of photochemical reduction of nitro blue tetrazolium (hereinafter: NBT) resulting in the blue reduction product of NBT with O2–. Solution in test tubes were stirred for few seconds and set in front of the light source for 10 minutes (Auclair and Voisin, 1985). The unit of SOD was the quantity of enzyme that inhibited NBT reduction by 50 % at 25 °C and 560 nm. All absorbance reading for content of soluble proteins, SOD and FRAP were done using Janway UV / VIS spectrophotometer 6505 and for MDA reading, Multiscan Spectrum Thermo Corporation.