2.3.4 Hormone content measurement in Exp. 3
Four grains from each treatment in Exp. 3 were sampled at DAP 16, 32 d after vitro culture, then frozen in liquid nitrogen for 2 min and stored at -80℃ for GA3, ZR, IAA, ABA measurements. The methods for hormones measurements from those described by Zhang et al. 2009, four frozen grains removed embryos were ground in an ice-cold mortar in 5 mL 80% (V/V) methanol extraction medium containing 1mM BHT as an antioxidant. The extract was incubated at 4℃ for 4 h and centrifuged at 5000 g for 15 min at the same temperature. The supernatants were passed through Chromosep C18 columns (C18Sep-Pak Cartridge, Waters Corp, Millford, MA, USA), pre-washed with 10 mL 100% (V/V) and 5 mL 80% (V/V) methanol, respectively. About 5 mL purified fraction was collected and dried under N2, and was dissolved in 1.5-3.0 mL phosphate buffer saline containing for analysis by an enzyme-linked immunosorbent assay (ELISA), which was provided by China Agricultural University.