title: Laser Capture Microdissection Protocol for Xylem Tissues of Woody Plants.
abstract: Laser capture microdissection (LCM) enables precise dissection and collection of individual cell types from complex tissues. When applied to plant cells, and especially to woody tissues, LCM requires extensive optimization to overcome such factors as rigid cell walls, large central vacuoles, intercellular spaces, and technical issues with thickness and flatness of the sections. Here we present an optimized protocol for the laser-assisted microdissection of developing xylem from mature trees: a gymnosperm (Norway spruce,Picea abies) and an angiosperm (aspen,Populus tremula) tree. Different cell types of spruce and aspen wood (i.e., ray cells, tracheary elements, and fibers) were successfully microdissected from tangential, cross and radial cryosections of the current year's growth ring. Two approaches were applied to achieve satisfactory flatness and anatomical integrity of the spruce and aspen specimens. The commonly used membrane slides were ineffective as a mounting surface for the wood cryosections. Instead, in the present protocol we use glass slides, and introduce a glass slide sandwich assembly for the preparation of aspen sections. To ascertain that not only the anatomical integrity of the plant tissue, but also the molecular features were not compromised during the whole LCM procedure, good quality total RNA could be extracted from the microdissected cells. This showed the efficiency of the protocol and established that our methodology can be integrated in transcriptome analyses to elucidate cell-specific molecular events regulating wood formation in trees.
authors.affiliation: Viikki Plant Science Centre, Department of Biosciences, University of Helsinki Helsinki, Finland.
authors.forename: Olga
authors.initials: O
authors.lastname: Blokhina
authors.affiliation: Plant Stress Signaling, Instituto Gulbenkian de CiênciaOeiras, Portugal; Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural SciencesUmeå, Sweden.
authors.forename: Concetta
authors.initials: C
authors.lastname: Valerio
authors.affiliation: Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural SciencesUmeå, Sweden; Department of Plant Developmental Biology, Institute of Experimental Biology, University of WrocławWrocław, Poland.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Sokołowska
authors.affiliation: Viikki Plant Science Centre, Department of Biosciences, University of Helsinki Helsinki, Finland.
authors.forename: Lei
authors.initials: L
authors.lastname: Zhao
authors.affiliation: Viikki Plant Science Centre, Department of Agricultural Sciences, University of Helsinki Helsinki, Finland.
authors.forename: Anna
authors.initials: A
authors.lastname: Kärkönen
authors.affiliation: Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences Umeå, Sweden.
authors.forename: Totte
authors.initials: T
authors.lastname: Niittylä
authors.affiliation: Viikki Plant Science Centre, Department of Biosciences, University of Helsinki Helsinki, Finland.
authors.forename: Kurt
authors.initials: K
authors.lastname: Fagerstedt
keywords: RNA integrity; cryosection; laser capture microdissection; ray cells; tracheids; xylem fibers