Seed components and nutritional evaluation in Zn and Fe biofortified bread wheat

Certified seeds of Triticum aestivum L. cv Nabão (0Ts) were sown in a walk-in growth chamber, under environmental controlled conditions (80% RH; 24/20ºC day/night temperatures; PPFD of ca 700 μmol m-2 s-1, 12 h photoperiod). Pots were irrigated with a standard solution (1Ts), during 1 month following germination. Until harvest, half of the pots were irrigated with 1Ts solution and the others with 5 fold nutrients (1T5s). After germination of 1T5s seeds, five treatments were established according to nutrients concentration: 2Ts (standard solution), 2T5s, 2T7s, 2T10s and 2T13s (5, 7, 10 and 13 fold nutrients, respectively). Carbohydrates were extracted with water, identified and quantified by HPLC, using standards of raffinose, sucrose, glucose and fructose. Starch was quantified by polarimetry. Organic matter was determined by incineration at 550 ºC for 2 h in a muffle furnace and ash by incineration, followed by nitric acid digestion. Nitrogen content was determined by the Kjeldahl method and protein by multiplying nitrogen content for a factor of 5.38. It was found that starch, raffinose and sucrose varied inversely with the amount of iron and zinc applied, while the levels of glucose and fructose varied in direct ratio of the concentrations (probably due to hydrolysis of starch and inversion of sucrose and raffinose). Ash and protein content increased, while organic matter decreased slightly Total lipids were extracted from grains using n-hexane. Samples were saponified, methylated and analysed by GC. Fatty acids were identified by means of known standards. Total fatty acids strongly decreased in 2T10s, indicating less abundant lipids. In the remaining treatments values were similar to control (0Ts) except in 2Ts, which showed slightly lower amounts. The most abundant fatty acid (FA) was linoleic (C18:2), followed by palmitic (C16:0), oleic (C18:1), linolenic (C18:3) and stearic (C18:0), and their relative distribution was not altered by nutrients increases. C18:2 and C18:1 decreased in all treatments, while higher C16:0 occurred in 2Ts and 2T5s. C18:3 was unaltered in 2Ts, and increased with higher nutrients concentrations. Lower C18:0 occurred in 2Ts and 2T5s. Unsaturation decreased in all treatments, particularly in 2Ts and 2T5s. The distribution of Zn, Fe, P, Ca, K, Mg, Cu and Mn was studied throughout a transect in the grain endosperm using Scanning Electron Microscopy coupled with a X-ray Microanalyser. SEM observations were complemented with AFLP markers studies, to evaluate genetic homogeneity at DNA level. It was concluded that in the large majority of the cases a distribution of the nutrients throughout the grain endosperm (between Periphery and Centram area) is not homogeneous, varying according to the element and the plant treatment. When analysing the ratio Fe/Zn a progressive increase until the 27Ts treatment was verified (0Ts - 0.70; 2Ts - 0.88; 2T5s - 0.98; 2T7s - 1.48), decreasing after that (2T10s - 1.10; 2T13s - 0.67), what partially agrees with the Fe/Zn ratios recently obtained by a member of this team using Atomic Absorption Spectrophotometry.