Lipid peroxidation was estimated by the level of malondialdehyde (MDA) production by a slight modification of the thiobarbituric acid (TBA) method described by Buege and Aust (1978). Leaves (0.25 g) samples were homogenized with a mortar and pestle in 5 mL of 0.1% (w/v) trichloroacetic acid (TCA) solution. The homogenate was centrifuged at 15 000 x g for 15 min and 1.0 ml of the supernatant was mix to the 4 ml 0.5 % (w/v) TBA in 20% TCA (w/v) then heated in a boiling water bath for 30 min and allowed to cool in an ice bath. The resulting supernatant was used for spectrophotometric determination of MDA. Absorbance at 532 nm was recorded and corrected for nonspecific absorbance at 600 nm. MDA concentrations were calculated by means of an extinction coefficient of 156 mM-1 cm-1 and the following formula: MDA (µmol/g fresh wt.) = [(A532 - A600)/156] x 103 x dilution factor (Zhanyuan and Bramlage, 1992).
Catalase activity was estimated by the method of Cakmak and Horst (1991). The reaction mixture contained 100 µl crude enzyme extract, 200 µl 10 mM H2O2 and 700 µl 50 mM potassium phosphate buffer. The decrease in the absorbance at 240 nm was recorded for 3 min by spectrophotometer, model Cintra 6 GBC (GBC Scientific Equipment, Dandenong, Victoria, Australia). CAT activity of the extract was expressed as CAT units per milligram of PROT.
Superoxide dismutase activity was determined according to the method of Giannopolitis and Ries (1997). The reaction mixture contained 100 µl 1 lM riboflavin, 100 µl 12 mm l-methionine, 100 µl 0.1 mm EDTA (pH 7.8), 100 µl 50mm Na2Co3 (pH 10.2), and 100 µl 75 lM nitroblue tetrazolium (NBT) in 2300 µl 25mm sodium phosphate buffer (pH 6.8), with 200 µl crude enzyme extract in a final volume of 3 ml. SOD activity was assayed by measuring the ability of the enzyme extract to inhibit the photochemical reduction of NBT. Glass test tubes containing the mixture were illuminated with a fluorescent lamp (120 W); identical tubes that were not illuminated served as blanks. After illumination for 15 min, the absorbance was measured at 560 nm. One unit of SOD was defined as the amount of enzyme activity that was able to inhibit by 50 % the photoreduction of NBT to blue formazan. The SOD activity of the extract was expressed as SOD units per milligram of PROT.
Ascorbate peroxidase activity was measured according to Nakano and Asada (1981). Tissue was homogenized in 1 mL of 50 mM phosphate buffer (pH 7.0) containing 5 mM ascorbate, 5 mM DTT, 5 mM EDTA, 100 mM NaCl and 2% (w/v) polyvinyl pyrrolidone (PVP). The homogenized material was centrifuged at 15,000 Ã- g for 15 min at 4ËšC. The reaction was initiated by adding 200 μl H2O2 10 mM to a final concentration of 44 μM as described by Nakano and Asada (1981). The reaction rate was monitored by decrease in absorbance at 290 nm.
Glutathione reductase activity was determined by the method of Halliwell and Foyer (Halliwell and Foyer, 1978). Its activity was assayed in a 1 mL reaction mixture containing 0.25 mL of 100 mM potassium phosphate buffer (pH 7.0), 0.05 mL of 10 mM oxidized glutathione, 0.12 mL of 1mM NADPH, 0.48 mL of distilled water, and 0.1 mL of enzyme extract. The resultant decrease in NADPH was observed at 340 nm.
Peroxidase activity was determined by the oxidation of guaiacol in the presence of H2O2. The increase in absorbance was recorded at 470 nm (Ghanati et al. 2002). The reaction mixture contained 100 μL crude enzyme, 200 μL H2O2 10 mM, 200 μL guaiacol 28 mM and 500 μL potassium phosphate buffer 50 mM (pH 7.0). Peroxidase activity of the extract was expressed as μmol tetraguacial min-1 mg-1 protein.
Proline assay
Proline (PRO) content was determined according to the method of Bates et al. (1973), which was modified as follows. Samples of leaves (0.2 g) were homogenized in a mortar and pestle with 3 ml sulphosalicylic acid (3 % w/v), and then the homogenate was centrifuged at 18,000 g for 15 min. Two millilitres of the supernatant were added into which 2 ml glacial acetic acid and 2 ml freshly prepared acid ninhydrin solution (1.25 g ninhydrin dissolved in 30 ml glacial acetic acid and 20 ml 6 m orthophosphoric acid) were added. Tubes were incubated in a water bath for 1 h at 100 °C, and then allowed to cool to room temperature. Four millilitres of toluene were added and mixed on a vortex mixer for 20 s. The test tubes were allowed to stand for at least 10 min to allow the separation of the toluene and aqueous phases. The toluene phase was carefully pipette out into a glass test tube, and its absorbance was measured at 520 nm in a spectrophotometer. The content of PRO was calculated from a PRO standard curve and was expressed as mmol g)1 per fresh weight.
Photosynthetic and gas exchange
Measurements
Photosynthetic parameters, PN, gS and E were recorded on fully expanded leaves of second youngest nodes at 30 days after sowing using an infra-red gas analyzer (IRGA, LICOR, 6200, Lincoln, NE, USA) between 11:00 and 13:00 h at light saturation intensity. These observations were recorded on five plants in a treatment.
PRESS
UV-B absorbing pigments (flavonoids and anthocyanins) were extracted fromfresh leaf samples using the method of Jordan et al (1994). Fresh leaves were ground to a powder in liquid nitrogen. The flavenoids and anthocyanins were extracted in 10 ml of acidified methanol (79:20:1, v/v, methanol: water: HCL). Absorption spectra of the extracts were spectrophotometricaly determined and flavonoid and anthocyanin concentrations were estimated from absorption intensities (Ab) at 300 and 530 nm, respectively (Nogués et al. 1998).
Determination of anthocyanin and pheophytin Contents
The relative amount of anthocyanin was estimated spectrophotometrically after extraction in acidified methanol (methanol:water:HCl: 79:20:1) as described by Mancinelli (1984) using A530 - 0.25A657 to correct for Chl and non-specific degradation products.
For determination of pheophytin, leaf samples were extracted in 80% acetone and the percentage of Chl converted to pheophytin was estimated by an increase in absorbance at A553 relative to the absorbance at A665 (Bowler et al., 1991).
Determination of H2O2 content
Hydrogen peroxide levels were measured according to Velikova et al. (). Leaf tissues (0.5 g) were homogenized in ice bath with 5 ml 0.1% (w:v) TCA. The homogenate was centrifuged at 12 000 g for 15 min. 0.5 ml of the supernatant was added to 0.5 ml 10 mM phosphate buffer and 1 ml 1 M KI. The absorbance of supernatant was read at 390 nm. The content of H2O2 was given on a standard curve.
7Chlorophyll fluorescence
Chlorophyll fluorescence parameters (PSII maximum efficiency, Fv/Fm) of leaves of 5 plants for each group were measured by Plant Efficiency Analyzer of Hansatech, UK.
Relative water content
Five leaf discs were obtained from plants in every group on days 0 and 7 of salinity treatment. After FW of these discs were determined, they were floated on deionized water for 7 h under low irradiance. Two turgid tissues were then quickly blotted dried prior to determining turgid weight. DW was then determined after oven drying at 70_C for 72 h, the time point at which a constant weight was reached. The RWC was calculated using the following formula (Smart and Bingham 1974):
RWC= (FW-DW/turgid weight-DW)*100
Pigments Chlorophyll and carotenoid
Photosynthetic pigments
Photosynthetic pigments in new fully expanded leaves from each treatment were extracted using 0·02 g of fresh material in 10 mL of 100 % acetone and kept in 4C for 24 hours. The suspension was centrifuged, and chlorophyll a (Chl a), chlorophyll b (Chl b) and carotenoids (Cx + c) contents were determined with a Hitachi U-2001 spectrophotometer (Hitachi Ltd, Japan), using three wavelengths (663·2, 646·8 and 470·0 nm). Concentrations of pigments [µg g fresh weight (f. wt)-1] were obtained by calculation, using the method of Lichtenthaler (1987).
Plant material
Chinese broccoli cultivares …. Were used in this study, which was carried out using a hydroponic system under green house conditions. Maximum and minimum temperatures ranged between …… and …. respectively . humidity was between ……… and photosynthetic photon flux density was ca. ……..
Chinese broccoli cultivars were subjected to 0, 25, 50, 75 and 100 mM NaCl concentrations in a nutrient solution, with four replications per treatment, giving a total of … experimental units (cultivar x NaCl concentrations x replications) , which were arranged in a randomized complete block design. This experiment was conducted from ……..
Hydroponic system
Salinity experiments were conducted in a closed loop hydroponic system, which allowed for recirculation of nutrient solution. The system was composed of three independent sections, each one having its own pump () and a 40 L container. Each section managed a single NaCl concentration (0, 50 and 100 mM) in the nutrient solution, and fed a total of 12 trays, which corresponded to three replications of that salt concentration for each cultivar. Trays were mounted on a temporary structure over a bench, while pumps and nutrient solution containers were seated on concrete blocks placed on the floor.
A simplified diagram of the hydroponic system used is shown in figure …. The diagram is out of scale, and it has been simplified to display a single section with main components.
Each pump in a single section delivered the nutrient solution from the container to the trays within that section through a 1.5 cm diameter irrigation hose. A piece of 0.5 cm diameter spaghetti tubing connected to this hose supplied the solution to each tray. A two-gph emitter was attached to the tubing tip to provide a uniform supply of nutrient solution to each tray. Trays were made of 40-cm 20 cm length, 10 cm height, and perlite was used as a mechanical support for seedlings as well as for nutrient solution retention.
Before being used for the experiments, perlite was washed and passed through a mm sieve to remove all small particles that could clog tubing and emitters in the system. Also, two cm circular pieces of plastic screen (mm mesh) were placed in the bottom inside of each tray to keep perlite from leaching and clogging the system.
A piece of spaghetti tubing connected to the bottom of each pot drained excess nutrient solution to a 1.5 cm diameter returning hose, which carried the solution back to their respective containers. Since the pumps capacity exceeded the nutrient solution volume required for the total number of pots being irrigated in a single section, a pressure - released valve was connected to the irrigation hose. Excess pressure was released by diverting part of the nutrient solution through this valve directly back to the container.
Because of perlite's capacity to retain nutrient solution for root uptake, there was no need to keep pumps running 24h a day. Hence, all four pumps in the system were plugged into a surge protector, and this in turn connected to a timer to turn pumps on for 60 min every other hour. It was observed on previous test that this on/off cycle was adequate to keep perlite moist all day long.
The seeds of four Chinese kale (Brassica alboglabra) cultivars, {"Kailan (11-33121)", "Hong Kong Kailan (22-N38005)" , "142 type B Chinese kale Curly Leaf" and "143 Hong Kong Stem Flower Chinese Kale"} were obtained from (Sin Seng Huat Seeds SDN BHD) and (Digistar Rauland MSC SDN.BHD). Seed's surface were sterilized and washed with water. The experiment was conducted in a green house at the University Putra Malaysia, Selangor, …… . Seeds were sown in seed trays and kept under water spray until emergence. Young seedlings in the seed trays were put in a film plastic pool for irrigating with a half strength Hoagland's nutrient solution with periodic loop irrigation. Seedlings were allowed to grow until 15th day after germination, and then they were transplanted and installed on the static hydroponic system and let them to growth for another one week, every tray contained twelve plants. At the seedling stage (21st day after germination) salt stress was started by adding appropriate amount of NaCl in the solution. Salinized solution included (control, 25 mM, 50 mM and 75 mM). The salt was added gradually to avoid osmotic shock. Two weeks after salt stresses were imposed; five plants of each pot were harvested. Leaf number, leaf area, the fresh and dry weight of leaf, shoot and roots of each plant were determined. After recording of fresh weights, the samples were dried in 65°C. Chlorophyll fluorescence, and relative water content were measured in plants before harvest and samples were obtained for further biochemical traits.
