An Efficient Antipodal Cell Isolation Method for Screening of Cell Type-Specific Genes in Arabidopsis thaliana

Abstract

In flowering plants, the mature embryo sac consists of seven cells, namely two synergid cells and an egg cell at the micropylar end, one central cell, and three antipodal cells at the chalazal end. Excluding the antipodal cell, as a model for the study of cell fate determination and cell type specification, the roles of these embryo sac component cells in fertilization and seed formation have been widely investigated. At this time, little is known regarding the function of antipodal cells and their cell type-specific gene expression patterns. One reason for this is difficulties related to the observation and isolation of cells for detailed functional analyses. Here, we report a method for antipodal cell isolation and transcriptome analysis. We identified antipodal cell-specific marker line K44-1, and based on this marker line, established a procedure allowing us to isolate antipodal cells with both high quality and quantity. PCR validation of antipodal-specific genes from antipodal cell cDNA showed that the isolated cells are qualified and can be used for transcriptome analysis and screening of cell type-specific marker genes. The isolated cells could keep viable for a week in culture condition. This method can be used to efficiently isolate antipodal cells of high quality and will promote the functional investigation of antipodal cells in Arabidopsis thaliana. This increases our understanding of the molecular regulatory mechanism of antipodal cell specification.

Two-Step Maternal-to-Zygotic Transition with Two-Phase Parental Genome Contributions

“…The Arabidopsis ovules in the upper one-third of siliques after 14, 24, 36, and 80 HAP were collected individually under a fluorescence microscope to isolate 14 and 24 HAP zygotes and 1-and 32-cell embryos, respectively. The isolation of egg cells and 14 HAP zygotes was performed according to our previous protocol with some modifications (Yu et al, 2016). Egg cells and 14 HAP zygotes were directly dissected manually from ovules in 13% mannitol solution under an inverse fluorescence microscope.…”