Laser Capture Microdissection for Amplification of Alternative Oxidase (AOX) Genes in Target Tissues in Daucus carota L

Laser microdissection provides a useful method for isolating specific cell types from complex biological samples for downstream applications. In contrast to the texture of mammalian cells, most plant tissues exhibit a cell organization with hard, cellulose-containing cell walls, large vacuoles, and air spaces, thus complicating tissue preparation and extraction of macromolecules such as DNA. In this study, we report a method that allows tissue-specific gene amplification. An improved perception of genetic identity of the entire plant can contribute to improved functional marker strategies. Alternative oxidase (AOX) has crucial position for stress-induced responses/adaptation. Daucus carota sequence polymorphisms in AOX were identified, however, never at tissue/cell level. This technology will support studying AOX gene sequences in carrot organs/tissues/cells and specifically exploring differential polymorphisms in root meristem that might be associated to adaptive growth upon all kind of stresses. Details on aspects of tissue preparation, including fixation and embedding procedures, laser capture microdissection, DNA extraction, and amplification, are provided. A combination of laser microdissection and polymerase chain reaction amplification provides a powerful tool for the analysis of AOX gene amplification in methacarn-fixed paraffin-embedded tissues.