2.3.1 Measurement and sampling in Exp. 1
Four maize plants were selected in each treatment at the tasseling stage. The maximum length (L, cm) and real width (W, cm) of each leaf were measured using the formula (L×W×0.75) to calculate the total plant leaf area. The plant tagged were measured 9 times on DAS (Days after sowing) 61, 66, 75, 82, 89, 99, 108, 118, 123. The SPAD-502 chlorophyll meter was used to monitor chorophyll concentrations (a.u., i.e., SPAD reading) in ear leaf of selected plant. The middle section of the ear leaf was measured 5 times, avoiding the main leaf vein on DAS 59, 64, 69, 75, 82, 89, 99, 108, 118, 123. The net photosynthetic rate of the ear leaf was measured at 10:00-11:00am using Li-CorLI-6400XT Portable Photosynthesis System (Licoln, NE, USA) at DAS 64, 69, 75, 82, 89, 99,108, 123.
Plants growing well were tagged and hand-pollinated by enclosing ears in bags from silk emergence until the last emerged silks became senescent. 200 superior grains (middle grains of spike) and 200 inferior grains (upper grains of spike) from above plants were obtained to determine the grain dry weight at DAS 78, 88, 98, 108, 118, 123. All grains were dried at 70℃ to constant weight and weighed. The processes of grain filling were fitted by Logistic growth equation (Figure 3) (Gao et al., 2017):
W=K/(1+ae-bt)
Where W is the kernel weight (mg); K is the final kernel weight (mg); t is the time after pollination; a, b are coefficients determined by regression. The onset time of grain filling peak stage (d): t1=(lna-1.316) b-1; The end time of grain filling peak stage (d): t2=(lna+1.316) b-1; The end of grain filling (d): t3=(lna+4.595) b-1; The slow-increase period (d): T1=t1; The fast-increase period (d): T2=t2-t1; The slight-increase period (d): T3=t3-t2; The mean grain filling rate (mg grain-1 d-1): V=K t3-1; Mean grain filling rate of slow-increase period (mg grain-1 d-1): V1=Wt1 T1-1; Mean grain filling rate of fast-increase period (mg grain-1d-1): V2=(Wt2-Wt1) T2-1; Mean grain filling rate of slight-increase period (mg grain-1 d-1): V3=(Wt3-Wt2) T3-1.