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<p>Plant material according to the current plant ontologies: Embryo-derived tissues: whole plant (PO:0000003) at the development stage 1.05-coleoptile emerged from seed (GRO:0007056); Seedling leaves: primary shoot (PO:0006341) at the developmental stage 2.02-first leaf unfolded (GRO:0007060) (Druka et al. 2006).</p>
<p>To obtain embryo-derived tissue, growth room#2, AN building, SCRI, with the standard laboratory bench positioned in the middle of the room was used to germinate sterilized seeds. Seeds were placed between three layers of wet 3MM filter paper in the 156 10 mm Petri plates. Thirty to fifty seeds per line (per Petri plate) were used. Germination was in the dark, 16 hours at 17 deg C and 8 hours at 12 deg C. After 96 hours, embryo-derived tissue (mesocotyl, coleoptile, and seminal roots) from three grains was dissected and flash frozen in the liquid nitrogen. Germination and collection was repeated two more times. Complete randomization of the Petri plates was done for each germination event. Tissues from all three germinations (collections) were bulked before RNA isolation. Three replicates of the parental cultivars were germinated for each collection.</p>
<p>To obtain seedling leaves, three Microclima 1000 growth chambers (Snijders Scientific B.V., Tilburg, Holland) were used for the experiment. Each cabinet accomodated 40 (13x13 cm) pots. Humidity was set to 70%, with light conditions for 16 hours light at 17C and 8 hours dark at 12C. The cycle started at 10 am with lights on. Light intensity was 337-377 mmol m-2 s-1, measured at the beginning of the experiment, 11 cm from the light source. Measurement was done using Sky Quantium light sensor at 15oC. Plants were placed 55 cm from the light source (from the bulb to the surface of the vermiculite). Ten sterilized seeds per pot were planted and 3 pots per genotype / per cabinet were used. After 12 days, leaf blade and sheath from 5-7 the same size plants was cut off, bulked and flash frozen in the liquid nitrogen.</p>
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