Patterns of Growth and Cell Death in Diploid Arabidopsis Cell Cultures with Synchronised Cell Cycle

Abstract

Arabidopsis thaliana is a model plant widely used for studying the molecular biology of different plant developmental processes. The use of cultured arabidopsis cells with synchronised cell cycles in such studies has been constrained by difficulties encountered in establishing rapidly dividing, finely dispersed, diploid cell cultures suitable for synchronisation. Here we discuss methods for establishing and maintaining such cultures, their cell cycle synchronisation and use in programmed cell death studies. Leaf callus produced the fastest growing cell suspensions in Murashige and Skoog (1962) media supplemented with 2,4-D (4.5 ?M), NAA (5.37 ?M), IAA (5.71 ?M), BAP (2.2 ?M) and 3% sucrose. The cultures were grown in the dark at 24°C, 110 rpm and 50 mm orbit. Late-exponential stage cells (10 mL) were subcultured at weekly intervals producing about 250 mg/ml fresh cells in a week. Filtering cells to reduce the size of cell clusters, concentrating cells by removing spent medium after allowing the cells to settle to the bottom of flask, and paying special attention to the selection of small to medium cell clusters during subculture are key in achieving finely dispersed cells with reduced cell cycle duration. Among several treatments tested, aphidicolin at 4 mg/l resulted in a complete block of the cell cycle at G1/S interphase. Flow cytometry revealed ca.78% cell cycle synchronisation in the cell population. Ethylene and mannose were used to induce cell death in the highly synchronised cultures. Addition of these compounds to cultures 3 h after release from the aphidicolin block (G2 phase) resulted in elevated cell death (as measured by Evan's blue staining), but the patterns differed for the two compounds. Northern analysis revealed that expression of PCD associated genes precedes cell death.