Rice seedling treatment and root growth assays
Rice seeds were placed in a Petri dish (diameter 15 cm) filled with deionized water to accelerate germination at 32 ℃. The germinating seeds were placed in a 96-well plate in a plastic container. Afterwards, they were cultured in complete medium prepared according to the method described by the International Rice Research Institute. Every 1 L of culture solution (pH 5.8) contains 66.07 mg (NH4)2SO4, 93.6 mg NaH2PO4·2H2O, 101.1 mg KNO3, 33.37 mg CaCl2, 122 mg MgCl2·6H2O, 19 mg EDTA-Fe, 2.17 mg MnSO4·5H2O, 0.024 mg Na2MoO4·2H2O, 3.01 mg H3BO3, 0.2008 mg ZnSO4·7H2O, and 0.075 mg CuSO4·5H2O. Five-day-old seedlings of OE, knockout, and NIP were planted in a plastic plate with holes that were at a density of 10 × 10 cm (10 cm spacing between plants and 10 cm spacing between rows), and cultivated in a plastic box (110 cm × 90 cm × 50 cm). Eight rows and ten seedlings were arranged for each line, and five plastic boxes were designed for the test. The culture solution was replaced every 3 days. Seedlings were cultured in a greenhouse for 14 h at 32 °C, and at night for 10 h at 27 °C. Ten biological replicates were used for each treatment. The main root length of the rice seedlings was measured using a root scanner (WinRHIZO, Canada) at 5, 10, 15, 20, 25, and 30 days after seed germination. Adult plants (75 days old) were carefully separated from the soil of the field, and the roots were cleaned. The main root length was measured using a millimeter-scale ruler.