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We used wt plants (Oryza sativa L. ssp. ja … We used wt plants (Oryza sativa L. ssp. japonica ‘Nipponbare’), the control line FOX3, and transgenic lines transformed using the high-speed Agrobacterium-mediated transformation system (Toki et al., 2006).
qRT–PCR analysis of the T0 generation of 19 independent transformant lines revealed high LBD37/ASL39 expression in the RK16331–4 and RK16331–13 transformant lines; therefore, these lines were selected for further analysis.
The samples for the metabolite- and transcript-profiling experiments and the qRT–PCR experiments were prepared using the following procedure. The husks were separated from the seeds, and the brown rice seeds were sterilized by treating them with 70% ethanol for approximately 20 s. Subsequently, the seeds were stirred in 10 ml of sterilizing solution for 20 min by using an agitator (Rotator RT-50; Taitech Co., Ltd, Japan). After sterilization, the seeds were washed four times in 10 ml of distilled water by using the agitator. The sterilized seeds were inoculated on a solid half-strength MS medium containing 3% sucrose and 50 mg l−1 hygromycin (pH 5.8). After a 4-d incubation under dark conditions at room temperature, the plates were transferred to the growth chamber (MLR-350h/MLR-350 H; Sanyo, Japan). The following conditions were used for growth: 12 h of light at 30°C and 12 h of darkness at 25°C; relative humidity, 40%; light strength, 200–250 μmol s−1. Rice seedlings with a shoot length of 6–8 cm were hydroponically grown in half-strength hydroponic-culture solution (Makino et al., 1983) in a greenhouse (12 h of light at 30°C and 12 h of darkness at 25°C) for a week. Then, the seedlings were grown in a normal hydroponic culture solution in the greenhouse until the generation of the eighth leaf blade, after which the fifth and sixth leaves were harvested, bulked, and used for metabolite profiling, qRT–PCR, and transcriptomic analysis. The ages (DAS) of the RK16331–4 and RK16331–13 LBD37/ASL39-overexpressor plants and the controls differed at the time of sampling: the plants of lines RK16331–4 were sampled between 48 and 55 DAS; plants of lines RK16331–13, between 41 and 55 DAS; and the control plants, between 31 and 51 DAS. the control plants, between 31 and 51 DAS.
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