Laser Microdissection of Specific Stem-Base Tissue Types from Olive Microcuttings for Isolation of High-Quality RNA.

Abstract

Simple SummaryOnly a small portion of the stem cells participate in the process of adventitious root formation and the cells/tissues types involved in this process is species-dependent. In olive, it is still unclear which type of cells acquire competence for rooting. Regardless, the entire stem nodal segment (containing a mixture of distinct cell types) continues to be used in studies related to the molecular mechanisms underlying this process. Laser microdissection (LM) technology has been applied to isolate specific tissue and cell types. However, it is difficult to find a standard LM protocol suitable for all plant species and cell types and, thus, LM procedures must be developed and optimized for each particular tissue. In this study, we aimed to evaluate the efficiency of a LM protocol in olive microcuttings stem-base samples. This work presents a simple, rapid and efficient LM procedure for harvesting specific tissue types used for further high-quality RNA isolation. This will encourage future cell type-specific transcriptomic studies, contributing at deciphering rooting-competent cells in olive stems and to better understand the molecular mechanisms underlying the process of adventitious root formation.Higher plants are composed of different tissue and cell types. Distinct cells host different biochemical and physiological processes which is reflected in differences in gene expression profiles, protein and metabolite levels. When omics are to be carried out, the information provided by a specific cell type can be diluted and/or masked when using a mixture of distinct cells. Thus, studies performed at the cell- and tissue-type level are gaining increasing interest. Laser microdissection (LM) technology has been used to isolate specific tissue and cell types. However, this technology faces some challenges depending on the plant species and tissue type under analysis. Here, we show for the first time a LM protocol that proved to be efficient for harvesting specific tissue types (phloem, cortex and epidermis) from olive stem nodal segments and obtaining RNA of high quality. This is important for future transcriptomic studies to identify rooting-competent cells. Here, nodal segments were flash-frozen in liquid nitrogen-cooled isopentane and cryosectioned. Albeit the lack of any fixatives used to preserve samples’ anatomy, cryosectioned sections showed tissues with high morphological integrity which was comparable with that obtained with the paraffin-embedding method. Cells from the phloem, cortex and epidermis could be easily distinguished and efficiently harvested by LM. Total RNA isolated from these tissues exhibited high quality with RNA Quality Numbers (determined by a Fragment Analyzer System) ranging between 8.1 and 9.9. This work presents a simple, rapid and efficient LM procedure for harvesting specific tissue types of olive stems and obtaining high-quality RNA.