Fabrication of microcage arrays to fix plant ovules for long-term live imaging and observation

Abstract

The long-term observation of plant ovules is important to investigate embryogenesis and the development occurring inside. However, it has been difficult to observe the ovule consistently in a specific position because ovules are randomly dispersed on a substrate with conventional methods. In this work, we fabricated PDMS microcage arrays to fix plant ovules from Arabidopsis thaliana and observed them over a long period. Microcages were designed based on experimental data of ovule growth so that ovules could be positioned inside the cage and observed in real time. First, we performed the sieving test to investigate the relationship between ovule size and different microcage widths. Next, we performed ovule culture for a week using microcage arrays developed based on the results of sieving tests. We successfully positioned and fixed ovules inside the microcages and confirmed that the ovules did not move from their original positions even though the culture dish was frequently moved for observation and incubation. Moreover, we observed that the ovules grew inside the cages without being affected due to the flexibility of PDMS. Thus, we confirmed the validity of microcage arrays as the useful tool to fix and culture plant ovules for long-term observation. We expect that microcage arrays could be widely used as the essential device for pursuing scientific findings in further researches of plant embryogenesis and reproduction.