- Institutionen för skoglig genetik och växtfysiologi, Sveriges lantbruksuniversitet
- Instituto Gulbenkian de Ciência
Blokhina, Olga; Valerio, Concetta; Sokolowska, Katarzyna; Zhao, Lei; Karkonen, Anna; Niittyla, Totte; Fagerstedt, Kurt
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 years 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.
cryosection; laser capture microdissection; ray cells; RNA integrity; tracheids; xylem fibers
Frontiers in Plant Science
2017, Volym: 7
Utgivare: FRONTIERS MEDIA SA