2.1 Plant materials and growth conditions
Seeds of Nipponbare, a japonica rice (Oryza sativa ) cultivar, were purchased from Nouken Inc. (Kyoto, Japan). Transgenic rice lines overexpressing bacterial biuret hydrolase, which were generated from Nipponbare in this study, were self-pollinated to obtain T1 progenies. T1 and T2seeds were used for experiments.
Rice plants were grown under hydroponic conditions in a growth chamber (NS-280 FHW; Takayama Seisakusyo, Kyoto, Japan) under a temperature, photo period, and light intensity of 30ºC, 12 h, and 350 mmol m-2 s-1, respectively. Rice seeds were soaked in distilled water supplemented with fungicide (Trifumin; Nippon Soda Co., Ltd., Tokyo, Japan) for three days at 30ºC. Ten or twelve of the imbibed seeds were sown on a nylon-mesh (18 mesh, 24 × 36 mm) supported by a plastic frame floating on the culture solution. The culture solution contained 1 mmol L-1(NH4)2SO4, 0.25 mmol L-1 KH2PO4, 0.5 mmol L-1 KCl, 0.5 mmol L-1CaCl2, 0.5 mmol L-1MgCl2, 0.09 mmol L-1ethylenediamine-N,N,N’,N’-tetra acetic acid, iron(III), sodium salt, trihydrate (FeNa-EDTA) and Arnon’s micronutrient (cited by Hewitt, 1966). The culture solutions were not aerated, and the solutions were renewed once a week. Seeds that did not germinate were removed from the mesh at an appropriate time. Biuret was included in the culture solution whenever necessary.