Trichome isolation with and without fixation using laser microdissection and pressure catapulting followed by RNA amplification: Expression of genes of terpene metabolism in apical and sub-apical trichome cells of Artemisia annua L.

The aim of this project was to evaluate the effect of fixation on plant material prior to Laser Microdissection and Pressure Catapulting (LMPC) and to identify an appropriate method for preserving good RNA quality after cell isolation. Therefore, flower buds from Artemisia annua L. were exposed to either the fixative formaldehyde or a non-fixative buffer prior to cell isolation by LMPC. Proteinase K was used after cell isolation from fixed plant tissue, in an attempt to improve the RNA yield. The ability to detect gene expression using real-time quantitative PCR with or without previous amplification of RNA from cells isolated by LMPC was also evaluated. Conclusively, we describe a new technique, without fixation, enabling complete isolation of intact glandular secretory trichomes and specific single trichome cells of A. annua. This method is based on LMPC and preserves good RNA quality for subsequent RNA expression studies of both whole trichomes, apical and sub-apical cells from trichomes of A. annua. Using this method, expression of genes of terpene metabolism was studied by real-time quantitative PCR. Expression of genes involved in artemisinin biosynthesis was observed in both apical and sub-apical cells.

► Evaluated the effects of fixation on plant material.
► Developed a method without fixation for cell isolation by LMPC.
► Preserved good RNA quality for subsequent RNA expression studies by qPCR.
► Evaluated the need for amplification of RNA after LMPC.
► Concluded that artemisinin biosynthesis occurs in both apical and sub-apical cells.