Laser microdissection on Norway spruce bark tissue: A suitable protocol for subsequent real-time reverse transcription–polymerase chain reaction (RT-PCR) analysis

Abstract

The development of new tools able to select specific plant tissue is crucial for gene expression studies. During the last years, the use of laser microdissection, mainly tested on herbaceous plant tissue, has been found to be a useful technique for these purposes. This method is poorly tested on woody species, and so far no studies of gene expression have been applied on forest trees. For this reason the present work proposes the optimization of a functional protocol using laser microdissection pressure catapulting (LMPC) and real-time reverse transcription–polymerase chain reaction (RT-PCR) in bark stem tissue of Norway spruce (Picea abies). Bark tissue fragments were collected from Norway spruce trees and sliced with a cryostat. RNA was extracted from both whole cross-sections and microdissected bark cells. The feasibility of the method was confirmed by the amplification of the α-tubulin, an endogenous gene of P. abies, with efficiency comparable to that obtained from non-microdissected tissue. The proposed protocol, here adapted for bark tissue of woody species, represents a useful tool in a wide range of hosts that, unlike herbaceous plants, have scarcely been considered up to now.

Keywords:

• α-Tubulin gene
• bark
• laser microdissection pressure catapulting
• Picea abies
• RT-PCR
• woody plants

Acknowledgements

This study was carried out by grants from the University of Florence ‘Fondi di Ateneo’. We gratefully acknowledge the Section of Plant Biology (DiBA, University of Florence) for cryostat sectioning.