*4.3. Determination of Seed Mineral Concentration*

Samples of dried seeds were finely ground using a vibrating sample mill (TI-100, Heiko, Japan) and the mineral concentrations of the seed were measured. Finely ground samples were digested by Sulfuric Acid-Hydrogen Peroxide (H2SO4–H2O2), and the K concentration was measured using a

flame photometer (ANA 135, Tokyo Photoelectric, Tokyo, Japan). Ca and Mg were measured using an atomic absorption flame emission spectrophotometer (AA-6200, Shimadzu, Japan). The total P was determined using a UV-Spectrophotometer (U-3310, Hitachi Co. Ltd. Tokyo, Japan) following the molybdenum reaction solution method [45]. The total nitrogen was measured using the Kjeldahl method after sample digestion with concentrated H2SO4 and H2O2. The crude protein concentration was calculated based on converting the seed nitrogen to protein percentage by multiplying by a conversion factor of 6.25 [46].

### *4.4. Determination of Phytate P and Inorganic P Concentration*

Seed phytate P was measured according to the method by Raboy et al. [47], where aliquots of flour were extracted in extraction media (0.2 M HCl: 10% Na2SO4) overnight at 4 ◦C with shaking. Extracts were centrifuged, and phytate was obtained as a ferric precipitate and assayed for P calorimetrically using ammonium molybdate reaction reagent. Inorganic phosphorus was extracted in trichloroacetic acid (12.5%) + MgCl2 (2 mmol <sup>L</sup>−1) while stirring overnight, and Pi was measured using colorimetric methods [45]. The phytic acid concentration was calculated by multiplying phytic acid P values by 3.55 as described by Raboy and Dickinson [48].

### *4.5. Determination of Zn, Fe and Mn Concentration*

Determination the Zn, Fe, and Mn concentration, the seeds were digested by HNO3 and H2O2 (4:1) and measured using an inductively coupled argon atomic emission spectrometer (iCAP 6000, Thermo Fisher Scientific Inc. USA).
