Plant materials and growth conditions
Seeds of japonica rice (Oryza sativa ) cultivar Nipponbare were purchased from Nouken (Kyoto, Japan). Transgenic rice lines overexpressing bacterial biuret hydrolase were developed from Nipponbare (Ochiai et al., 2020), and the T3 generation was used in this study. Seeds of rice were soaked in distilled water added with fungicide (Trifumin; Nippon Soda, Tokyo, Japan) for two days. Ten seeds were sown on a mesh (18 mesh, 23 x 34 mm) stretched on a plastic slide mount and floated on a culture solution. The culture solution contained 1 mmol L-1(NH4)2SO4, 0.5 mmol L-1 KCl, 0.25 mmol L-1KH2PO4, 0.5 mmol L-1CaCl2, 0.5 mmol L-1MgCl2, and Arnon’s micronutrient (cited by Hewitt, 1966). Iron was supplied at the rate of 5 mg Fe L-1 as Ethylenediamine-N,N,N’,N’-tetraacetic acid iron(III) sodium salt. Biuret was added to the solution whenever necessary. The culture solution was prepared with tap water and not aerated. At most, six nets were floated in a 1-L plastic container. Plants were raised in a growth chamber (NS-280 FHW; Takayama Seisakusyo, Kyoto, Japan) under the following conditions: temperature, 30°C; photoperiod, 12 h; and light intensity, 350 μmol m-2 s-1.
The rice Oc cell suspension culture line (Baba et al., 1986) was provided by RIKEN BRC, participating in the National BioResource Project of the MEXT/AMED, Japan. The cells were maintained as described by Ochiai et al. (2020).