Laser-Assisted Microdissection to Study Global Transcriptional Changes During Plant Embryogenesis

Abstract

Next-generation sequencing platforms are able to provide a comprehensive overview of gene activity in a particular biological sample. When coupled to techniques that isolate specific cell types, or the RNA from specific cell types, NGS can provide a detailed and granular view of the transcriptional activity in an organ or tissue. Laser-assisted microdissection is a microscopy-based technique that allows the isolation of discrete cell types from their tissular context while maintaining RNA integrity. In this chapter we will present an overview of the LAM technique, and present the few works that have undertaken transcriptomic analyses of plant embryos. While zygotic embryos, embryogenic calli or somatic embryos from different plant species have been studied using microarrays or high-throughput sequencing, LAM-based transcriptomics has only been applied to the Arabidopsis zygotic embryo and seed.

A high-resolution gene expression atlas of the medial and lateral domains of the gynoecium of Arabidopsis

“…With the goal of elucidating which genes are tissue-specifically expressed during the development of the gynoecium, we used the laser-assisted microdissection (LAM) method (Chávez Montes et al, 2016) combined with RNA-Seq. We generated a highresolution expression map by collecting different tissues at two time points.…”

“…The inflorescence embedding protocol is based on the protocol described by Chávez Montes et al (2016). In summary, open flowers were removed from each main inflorescence.…”

“…One technique to isolate specific cell types from plant tissues and organs, is Laser-Assisted Microdissection (LAM) microscopy. This technique has been consolidated for collecting cell populations from their tissue context, regardless of their complexity (Chávez Montes et al, 2016;Day et al, 2005;Florez Rueda et al, 2016;Kerk et al, 2003;Wuest & Grossniklaus, 2014). Despite the fact that the LAM technique is also labor intensive, the advantage is, unlike other techniques, that it does not require the generation of marker lines, protoplasts, or the need for previous information on a large number of marker genes, except for knowing the morphology of the tissues to be collected, resulting in a large number of marker genes (Chávez Montes et al, 2016;Day et al, 2005;Florez Rueda et al, 2016;Wuest & Grossniklaus, 2014).…”