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Potato tuber slices and leaf discs were extracted with 80% ethanol-50 mM HEPES KOH, pH 7.4, at 80°C. The supernatant was used for enzymatic analysis of glucose, fructose and sucrose . For starch measurement, extracted plant material was homogenized in 0.2N KOH, and following incubation at 95°C was adjusted to pH 5.5 with 1N acetic acid. Starch was hydrolyzed with amyloglucosidase, and the released glucose was determined enzymatically. | 15701180_p30 | 15701180 | Determination of starch and soluble sugar contents | 4.108459 | biomedical | Study | [
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Frozen plant tissue (400 mg) was powdered in liquid nitrogen and extracted with methanol (4 ml per g -1 fresh weight), heated for 15 min at 70°C, and centrifuged . Samples were diluted with water to 50% methanol concentration and extracted with chloroform (1:1 v/v). A portion of the water phase was dried under vacuum and used for derivatisation . Ribitol was used as an internal standard added directly to the sample homogenate (30 μg g -1 fresh weight). | 15701180_p31 | 15701180 | Tissue extraction for catecholamine content measurement | 4.053822 | biomedical | Study | [
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The dried extracts were incubated in pyridine/methoxyamine (20 mg mL -1 ) at 37°C for 90 min and then acidic protons were derivatised with N-Methyl- N-trimethylsilyltrifluoroacetamide (MSTFA) at 37°C for 90 min. 2 μl of sample was used for analysis . A HP quadrupole mass spectrophotometer , combined with a gas chromatograph HP 6890 and autosampler (all Hewlett Packard, Germany) and equipped with 30 m HP – 5MS, fused silica capillary column, was used. Injection temperature was 230°C, with the interface set to 250°C and the ion source adjusted to 180°C. The carrier gas used was helium set at a constant flow rate of 1 ml min -1 . The temperature program was 5 min isothermal heating at 70°C, followed by a 5°C min -1 oven temperature ramp to 310°C and final 1 min heating at 310°C. The system was then temperature equilibrated for 6 min at 70°C prior to injection of the next sample. Mass spectra were recorded at 2 scan s -1 with an m/z 50 – 600 scanning range. The chromatograms and mass spectra were evaluated using the MSD ChemStation program (Hewlett Packard, Germany). As standards, dopamine, norepinephrine, normetanephrine, L-Dopa, tyramine and tyrosine (Sigma) were used. The recovery samples were spiked with dopamine, norepinephrine, normetanephrine, L-Dopa, tyramine and tyrosine; the estimated recoveries were 80, 93, 85, 95, 82, 90%, respectively. The following ions were used for quantification: ribitol 307; 319, L-Dopa 218; 267; 368, dopamine 174; 338; 426, norepinephrine 174; 355, 514, normetanephrine 174; 297; 456, tyramine 174; 264; 338, tyrosine 218; 280, 354. The amounts of catecholamines were determined from the ratio of peak areas of catecholamines to peak area of the internal standard (ribitol). | 15701180_p32 | 15701180 | GC – MS analysis | 4.252688 | biomedical | Study | [
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Tissue was harvested, weighed and immediately frozen in liquid N 2 . 0.5 g ± 0.1 g tissue was homogenised in a chilled mortar in 2 ml of an extraction buffer containing 30 mM HEPES-NaOH, pH 6.9, 10 mM DTT, 1 mM MgSO 4 , 0.5 mM EDTA, 0.5% (w/v) BSA and 0.5% (w/v) PVP at 4°C. The homogenate was centrifuged at 16000 g for 10 min and the supernatant desalted using Sephadex G-25 columns. Enzyme activities were determined using the following published methods; SPS ; AGPase ; SuSy, PK ; PGM ; starch synthase ; PGI, enolase ; UGPase ; hexokinase and starch phosphorylase as described by . | 15701180_p33 | 15701180 | Preparation and analysis of samples for enzyme activities | 4.097568 | biomedical | Study | [
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The t-tests were performed using the algorithm embedded into Microsoft Excel. The term significant is used when P < 0.05. | 15701180_p34 | 15701180 | 3.9. Statistical analysis | 2.201385 | biomedical | Study | [
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AS carried out the metabolic analysis of transgenic plants and drafted the manuscript. AŚ carried out the construction and selection of transgenic plants and performed the statistical analysis. JS conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript. | 15701180_p35 | 15701180 | Authors' contributions | 0.867881 | biomedical | Other | [
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