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.