Li 300

At transplant and three weeks after transplant (harvest), height from the substrate surface to the meristem, leaf area of two (seedling) or four (harvest) most recently fully expanded leaves (measured with LI-300; LI-COR Biosciences), stem diameter (harvest reps 2 and 3) at the base, and shoot fresh mass were recorded. Additionally, the number of branches >2.5 cm and node number (rep 2 and 3) were recorded at harvest. Tissue was placed in a forced-air oven maintained at 75°C for at least 3 d, weighed, and dry mass was recorded. Three weeks after transplant, the two most recent, fully mature leaves of five plants from each treatment were detached, frozen, and stored at -20°C until gas chromatography mass spectrometry (GCMS) analysis as reported in Walters et al. [25 (link)] from a method derived from [26 (link)] Schilmiller et al. Tissue was ground in liquid nitrogen, and compounds were extracted with methyl tert-butyl ether (MTBE) with a tetradecane internal standard. Samples were analyzed using an Agilent 7890A GC and single quadrupole MS with 5975C inert XL MS detector (Agilent, Santa Clara, CA). Compound concentrations were normalized to the sample internal standard and leaf dry weight, then quantified using the standard calibration curves of 1,8 cineole, eugenol, linalool, and methyl chavicol with a tetradecane internal standard (Millipore Sigma; St. Louis, MO).

At 53 days after sowing, plants were harvested, and the leaf angle, leaf number, total leaf area, root length, leaf and root fresh weights, and leaf and root dry weights were measured. Total leaf area was determined using a leaf area meter (LI-300; Li-Cor Inc., Lincoln, NE, USA), and the dry weights of leaf and root were measured after oven-drying at 80°C for more than 72 h.
As a measure of the nutrient quality, the ascorbic acid and nitrate contents in the outer and inner leaves (all leaves in each group of layers were cut into pieces, and the 1 g fresh samples were used for measurements) of plants in each treatment were quantified by using an RQ Flex plus reflectometer (Merck, Darmstadt, Germany), following the method of Pantelidis et al. (2007) (link).

Five plants with average tillering number of each plot were harvested at heading and maturity stages in 2017 and 2018. Plant samples were separated into green leaves, stems, senescent organs, and panicles, and their dry weights were determined after oven-drying at 105 °C for 30 min and then at 85 °C to constant weight to calculate total biomass. The oven-dried samples of stem, which included culms and leaf sheaths, were used for determination of non-structural carbohydrate (NSC) concentrations (mg g⁻¹ dry weight) according to the method reported previously by Fu et al. (2011) (link). The total mass of NSC stored in stems (g plant⁻¹), apparent transferred mass of NSC from stems to grains during grain filling (g plant⁻¹), and apparent ratio of transferred NSC from stems to grains (%) were calculated according to the method of Li et al. (2018a) (link). The total green leaf area of each plant was measured using a leaf area meter (LI-300, Li-Cor Inc., Lincoln, NE, USA) in 2018. The leaf area index (LAI) was calculated by the ratio of the total green leaf area of each plant to the area of the ground covered (17 cm × 25 cm). We also measured the length and width of the flag and second leaves on the main stems in 2017 and 2018.