*4.6. Seed Phosphorus Assay*

Seed inorganic P (Pi) levels were assayed qualitatively according to the micro-determination method developed in reference [46] with modifications. The qualitative assay was used for the

identification of the high-inorganic-P (HIP) phenotype. The seeds were transferred to 96-well plates and extracted in 0.4 M HCl solution (10 μL per mg sample) overnight at 4 ◦C. Aliquots of 10 μL of supernatant per each sample were used for Pi level determination, according to reference [4] with slight modifications [6]. The development of a blue color implied increased level of Pi (HIP), while colorless samples typified the WT levels of parent varieties (Figure S1).

Seed Pi levels were also quantitatively determined according to reference [47] in triplicates, as follows. Brown rice grains were ground into rice flour, and ~400 mg rice flour per sample were extracted in 12.5% ( *w*/*v*) TCA (trichloroacetic acid containing 25 mM MgCl2) by gentle shaking overnight at 4 ◦C. After centrifugation at 15,000× *g* for 10 min, the supernatants were used for Pi assay according to reference [2].

The PA content was determined for brown rice flour according to reference [48] by using a commercial assay kit (Megazyme, Irland) in triplicate. Briefly, ~1 g of brown rice grains was mixed with 20 mL of hydrochloric acid (0.66 M), stirred vigorously for 3 h at room temperature, and then centrifuged at 13,000 rpm for 10 min. Then, 0.5 mL of the supernatant was immediately neutralized by the addition of 0.5 mL of sodium hydroxide solution (0.75 M). The neutralized sample extract was subjected to an enzymatic dephosphorylation reaction procedure. The absorbances at 655 nm was determined using an ultraviolet spectrophotometer.

Total seed phosphorus and mineral elements were determined according to reference [49] in triplicate. Briefly, brown rice samples were digested in a microwave digestion system (Mars6, USA) at 160 ◦C for 40 min, using ~200 mg of sample in 7 mL of HNO3. The digested solution was concentrated at 140 ◦C for 2 h until less than 1 mL of solution was left and then brought to 30 mL with ultrapure water [50]. Total seed phosphorus and mineral elements were analyzed by Inductively Coupled Plasma–Mass Spectrometry (ICP–MS) (PerkinElmer, MA, USA).
