Abstract
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.
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Acknowledgments
This work was supported through a Marie Curie Fellowship to C.R. by the European Commission through the project AGRO-AMF-AOX within the program Industry-Academia Partnerships and Pathways (IAPP, FP7) coordinated by the EU Marie Curie Chair. C.R. appreciates having been hosted during the 2-month fellowship by Inoq GmbH (Germany). We acknowledge equipment funding from the Programa Operacional Regional do Alentejo (InAlentejo) Operation ALENT-07-0262-FEDER-001871 and also support through National Funds by FCT—Foundation for Science and Technology under the Project UID/AGR/00115/2013. This work was supported by the program POPH-Operational Program for Human Potential and “Fundo Europeu de Desenvolvimento Regional” (FEDER) funds through the Operational Program for Competitiveness Factors-COMPETE, and national funds through FCT under the strategic project PEst-C/AGR/UI0115/2011 and PEst-OE/AGR/UI0115/2014. We appreciate contribution on primer design, and support on gene isolation and data analysis by Amaia Nogales, Hélia Cardoso e Tânia Nobre. B.A.S. is thankful to the University of Évora for continuous invitations as Coordinating Investigator since 2008 in order to prolong running of the established EU Marie Curie Chair financed initially by the EC in the period from 2005 to 2008.
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Ragonezi, C., Arnholdt-Schmitt, B. (2017). Laser Capture Microdissection for Amplification of Alternative Oxidase (AOX) Genes in Target Tissues in Daucus carota L.. In: Jagadis Gupta, K. (eds) Plant Respiration and Internal Oxygen. Methods in Molecular Biology, vol 1670. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7292-0_21
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DOI: https://doi.org/10.1007/978-1-4939-7292-0_21
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