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

Abstract

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.

Keywords: OCT medium; PEN-membrane glass slide; RNA quality number; adventitious roots; cortex; cryosectioning; epidermis; phloem; single cell.

Figure 1

Plant material preparation for laser microdissection. (A) olive microcutting showing the cutting site of the stem nodal segment; (B) stem nodal segments in cryomolds containing OCT medium; (C) cryomolds containing the segments being frozen in liquid nitrogen-cooled isopentane; (D) cryosectioning; (E) cryosections on a PEN-membrane glass slide after dehydration and drying. Scale bars, 4 mm.

Figure 2

Cryosections of the stem nodal segments of olive microcuttings at the site of adventitious root formation before (A,C,E) and after (B,D,F) laser microdissection of cells from the phloem (A,B), cortex (C,D), and epidermis (plus hypodermis) (E,F) tissues. Pi, pith; Co, cortex; Ep, epidermis; Ph, phloem; X, xylem.

Figure 3

Cross sections of the stem nodal segments of olive microcuttings at the site of adventitious root formation showing a vascular bundle (Pi, pith; Co, cortex; Ep, epidermis; Ph, phloem; X, xylem). (A) Tissue cryosection prepared as described in the material and methods of the present work. (B) paraffin-embedded tissue prepared as described in Macedo et al. [29].

Figure 4

Integrity of the total RNA isolated after microdissection of the epidermis (plus hypodermis) (a–c), phloem (d–f) and cortex (g–i) from stem-base tissue cryosections, using the Fragment Analyzer system. The three graphics for each tissue corresponds to three technical replicates. The results are shown as an electropherogram showing two peaks and a digital gel showing two bands corresponding to the two ribosomal RNA subunits (18SrRNA and 28SrRNA). The concentration ([ ]) for each RNA sample is indicated as well as the corresponding RNA Quality Number (RQN).