Genomic Characterization of Transgenic Wheat (Triticum Aestivum L.) Using Fluorescence IN SITU Hybridization

Abstract

Fluorescence in situ hybridization (FISH) is a powerful tool to visualize specific DNA fragments in cultured cells, tissue sections or whole-mount preparations. The cytogenetic slides that serve as a target for the labeled DNA probe might be prepared using any approach suitable for obtaining cells with appropriate morphology for imaging and analysis. In mitotic metaphases, this technique allows to detect the presence of chromosomes and/or introgressive chromosomal segments. This study was conducted to identify the transgene copy number by FISH in transgenic wheat (Triticum aestivum L.) lines previously obtained and compared the accuracy of this methodology to the traditional Southern blot technique. We obtain chromosomal preparations and evaluated mitotic metaphases testing four FISH protocols on a set of two transgenic wheat lines. A total of 24 experiments (three experiments for two transgenes in each transgenic line, and the control line) were conducted. FISH analyses of individual transgenic events revealed multiple insertions of transgene fragments. The results obtained in this work showed that FISH allows determining the number of the transgene insertions in the genome of transgenic wheat plants in an accurate way and emerges as a viable alternative to replace the traditional technique of hybridization, Southern Blot.