Establishment of rapidly proliferating rice cell suspension culture and its characterization by fluorescence-activated cell sorting analysis

A rapidly growing and fine-textured cell line, NB2P, was established from Japonica rice cultivar Nipponbare and characterized in this study. Addition of casein enzymatic hydrolysate (2 g/L) and pectinase (0.005%) to the suspension medium resulted in a 2-fold-increased rate of cell growth and reduced aggregation. Remarkably, the medium and conditions described here resulted in growth leading to a 9-fold increase in fresh weight 7 d after subculture. High-quality, well-dispersed nuclei were obtained from this NB2P cell culture. Fluorescence-activated cell sorting (FACS) analysis of the isolated nuclei showed a clear separation of each cell cycle phase in both small- and large-scale preparations. On the basis of representative data from the nuclei fraction in the G1 phase. purity of the sorted and recovered nuclei was higher than 98%. The studies described here demonstrate that NB2P culture can be a powerful tool for studying many important plant processes, including DNA replication and cell cycle-related pathways.