Diseño de la base de datos

All samples were oven-dried at 60 °C for three days until a constant weight showed. Materials were milled to 2 mm by machine mill and oven dried prior to digestion to ensure complete dehydration. Tissue samples (~0.1 g) were placed into a digestion tube containing an anti-bumping granule to which the digestion acid (2 mL) was added and left for 2 h in the fumehood. Operational blank as well as in-house reference material from pooled dried canopy leaves of field-grown B.napus cultivar Temple were included in each digestion run. Prior to loading the digestion tubes (57 tubes) into the heating rack, 1 mL of hydrogen peroxide (H2O2, 30 % w/w) was added.

Digestion tubes were then loaded into the heating rack of the Gerhardt block digestion system and heated at 330°C for 45 m. An additional 1 mL of hydrogen peroxide (H2O2,

30 % w/w) was added after the tubes cooled. The tubes were then subjected to 330°C for 30 m, until the solution became clear. Afterwards, reverse osmosis water (R.O. water, 48.6 ml) was added to the tube and mixed well using a WhirliMixer (Fisons,

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UK). The solution was then transferred to a scintillation vial. This method was generated by Matthew Mitchell in the mineral analysis laboratory at Warwick Crop Centre (previously known as Warwick HRI) and adopted from Bradstreet (1965); Horneck and Miller (1998) and uses the same standard each time.

For Kjeldahl N, samples were analysed on the flow injection analyser after a 1:100 dilution was performed using FIAstar 5000 Analyzer (supplied by FOSS UK) connected to an auto sampling system (Sampler 5027, FOSS UK). The system is fully controlled by the FIAstar 5000 SoFIA software. After verifying the correct cassette and detector filters, M =590 nm (the wavelength at which all prepared standards and samples are measured), and R=720 nm (the reference standard wavelength for the internal calibration of the instrument) was installed. Using the SoFIA software the ammonium 0-5 mg/L method was selected and the absorbance of the ammonium indicator is checked. The absorbance is adjusted to be in the range of 300 - 500 mAU using dropwise addition of 0.01M sodium hydroxide or 0.01M hydrochloric acid to water depending on the value shown by the indicator. A calibration check is then followed.

The calibration standards were prepared as dilutions (0, 0.5, 1, 2 and 5 mg/L) of 1000 mg/L ammonium-N stock solution, (NH4)2SO4. The diluted samples were then moved

to 11.5 ml Rohren tubes and placed in the auto sampler and subjected to analysis. During the experimental runs a 1 mg/L calibration check was applied after every 10 samples. 60 samples were analysed in each run and a total of 1394 samples were analysed. The SoFIA software calculated the concentration of ammonium (mg/L) in each sample from which the total Kjeldahl Nitrogen concentration was calculated as

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mg/L. the percentage of N in plant tissue was then calculated using the following formula:

𝑁 % = N (mg. L−1) ∗ final diluted volume (ml)

dired weight of digested tissue (g) ∗ 104

The principle of this analysis is that a carrier stream of the sample which contains ammonium ions is injected into a stream of sodium hydroxide (NaOH) to form gaseous ammonia. The ammonia gas passes across a gas diffusion membrane into an indicator stream where it will react with an admixture of acid-base indicators. The resulting colour change could be monitored photometrically at 590 nm (Crompton, 2001).

2.3.6

Data analysis

Analysis of Variance (ANOVA) was performed to study the effect of the treatments; crop type, genotype and tissue, and the interaction between them on the concentration of Nitrogen (%DW) at both growth stages; GS 6.2/6.3 and harvest according to the model; [genotype x tissue] at the GS 6.2/6.3, [(croptype ̸ genotype) x tissue] at harvest and [growth stage x variety x tissue] to compare between the two growth stages. TukeyHSD analysis at 1 % significant level was used as a Post Hoc test when overall significant differences were observed. The proportion of total and partial variance in N concentration that attributes to each treatment factor was reported as omega squared (ω²) and partial omega squared (ω²_{p) and calculated following the equations below} (Olejnik and Algina, 2000):

ω² =𝑆𝑆𝑡𝑟𝑒𝑎𝑡𝑚𝑒𝑛𝑡 − 𝑑𝑓𝑡𝑟𝑒𝑎𝑡𝑚𝑒𝑛𝑡 𝑀𝑆𝑒𝑟𝑟𝑜𝑟

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ω²𝑝 = _{𝑆𝑆𝑡𝑟𝑒𝑎𝑡𝑚𝑒𝑛𝑡}𝑆𝑆𝑡𝑟𝑒𝑎𝑡𝑚𝑒𝑛𝑡 − 𝑑𝑓𝑡𝑟𝑒𝑎𝑡𝑚𝑒𝑛𝑡 𝑀𝑆𝑒𝑟𝑟𝑜𝑟_{+ (𝑁 − 𝑑𝑓𝑡𝑟𝑒𝑎𝑡𝑚𝑒𝑛𝑡) 𝑀𝑆𝑒𝑟𝑟𝑜𝑟}

Where SS is the sum of squares, MS is the mean of squares, df is the degree of freedom and N is the total number of observations. The variety mean value and standard error of the mean (SEM) were determined where n =3 at GS 6.2/6.3 and n =5 at harvest. Outliers in boxplot are data values that are 1.5 times the interquartile range (1.5 x IQR) from either end of the box. Pearson correlation analysis was conducted to determine the Correlation Coefficient (r) and to investigate relationship between the variables (between N and other 11 mineral elements across seven plant tissue types) using genotype means. The linear relationship was examined by Regression Analysis. The equation of line of the best fit (𝑦 = 𝑏 + 𝑎𝑥) and the coefficient of determination r2 for simple regression analysis and adjusted R2 for multiple regression analysis were all determined. All statistical analyses were performed using the R environment for statistical computing and graphics (version 3.3.2, 2016).

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2.4

Results

Total Kjeldahl N concentration [N] as a percentage of dried weight, (%DW) have been determined in different plant tissue types of root, stem and seeds during two different time points of plant growth among a number of Brassica napus L. accessions (14 and 30 for GS 6.2/6.3 and harvest, respectively) grown under low N supply (no N fertiliser) in a field experiment.