Nuclei release methods comparison for fresh leaves of rice (Oryza sativa) for efficient high throughput flow cytometry ploidy studies

Abstract

Flow cytometry trituration methods and the efficiency of lysis buffer solutions efficiency are compared in this study for extraction of nuclei from fresh leaves of rice (Oryza sativa). The rRazor blade sample trituration (chopping) procedure has been widely used to release nuclei from tissues in many plant species, together and combined with different lysis buffers in many plant species for low throughput analysis. AlternativelyIn contrast, the bead beating trituration method has been barely rarely been used for DNA ploidy determination, despite it being proposed as a less tedious alternative procedure to prepare nuclear suspensions when combined with Hanson's lysis buffer. In this study, bead beating was assessed in with rice young rice leaves and compared with the traditional chopping procedure. Each trituration method was combined with one of three nuclear isolation buffers (i.e. Hanson's buffer, Otto's buffers and LB01). Bead beating was applied for the first time using any all three of the assessed Hanson, LB01 and Otto's buffers, resulting in a rapid and suitable effective procedure for ploidy determination in rice fresh rice leaves ploidy determination. In addition, bead beating saved time and money, while reducing the exposure of the user to harmful substances. TIt is concluded that he best results were obtained when Hanson's nuclear isolation buffer was combined with the bead beating trituration method.