Evaluation of red fluorescent protein (DsRed) as alternative visual marker of genetic transformation in cassava (Manihot esculenta Crantz)

Abstract

Red fluorescent protein (DsRed) from reef coral was evaluated in comparison with green fluorescent protein (GFP) as a reporter gene for cassava transformation. Cassava friable embryogenic callus (FEC) was transformed with ERtargeted versions of DsRed and GFP constructs driven by the 35S cauliflower mosaic virus promoter. Efficiency of transformation was comparable for both visual marker genes at averages of 119 and 163 expressing plants recovered per cc of settled cell volume FEC for GFP and DsRed, respectively. High and uniform DsRed expression was observed at the single cell and proliferating callus stages, in somatic embryos and within organs of whole in vitro and greenhouse-grown plants in a manner similar to GFP. Plants expressing GFP and DsRed were robust and phenotypically normal with regard to growth, vigor, and formation of storage roots when grown in the greenhouse. Expression of marker genes within cross sections of petiole, woody stem, and storage roots from greenhousegrown plants was determined. The interference of phenolic compounds and chlorotic tissues characteristic of the signal from GFP-expressing tissues was not observed within tissues transgenic for DsRed. Tissues and plants co-expressing DsRed and GFP were produced by co-culturing FEC with a mixed Agrobacterium suspension carrying GFP and DsRed gene constructs or by re-transformation of an existing GFP transgenic line with DsRed. Re-transformation of GFPexpressing tissues was the more efficient method for production of GFP/DsRed stacked plants. Co-expression of both marker genes within the same transformation unit was easily visualized at their respective wavelength with the aid of appropriate filters thus validating their potential for coexpression studies.

Establishment of an efficient transformation and CRISPR/Cas9-mediated gene editing system in Chinese local planting cassava (Manihot esculenta Crantz) cultivar SC8

“…Based on the optimized transformation system, approximately 120-140 transgenic lines per mL SCV were regenerated for cassava SC8 in approximately 5 months. This transformation frequency is significantly higher than previous studies that used model cultivar 60444 and cultivars of TME14 and T200 (Taylor et al, 2012;Nyaboga et al, 2015;Okwuonu et al, 2015).…”

Use of red, far-red, and near-infrared light in imaging of yeasts and filamentous fungi

“…It is important to note that interference with autofluorescence of chlorophylls and other cell constituents may mask GFP fluorescence in above-ground photosynthesizing plant tissues (Zhou et al 2005 ; Berg and Beachy 2008 ). Meanwhile, the interference between chlorophylls and red fluorescent proteins can be minimalized by suitable filter sets (Jach et al 2001 ; Okwuonu et al 2015 ) or can be resolved by spectral unmixing techniques (Berg and Beachy 2008 ).…”

Vector backbone integration in transgenic cassava is significantly correlated to T-DNA copy number

“…The mother plants were maintained on Murashige & Skoog basal medium (MS2) (Murashige & Skoog, 1962) supplemented with 2% w/v sucrose (Taylor et al, 2012). The FEC target tissue was transformed with A. tumefasciens strain LBA4404 independently carrying p8016, p8052 and p900 constructs as described by Okwuonu et al (2015). Dot-blot analysis of transgenic plants DNA was extracted from leaf samples of randomly selected plant lines recovered from p8016, p8052 and p900 transformations using Qiagen DNeasy Plant Mini kit (CA, USA) following manufacturer's instruction.…”

“…The p8016 construct was designed using the MLB approach (Kuraya, 2004). We also incorporated a red fluorescent protein (DsRed) (Wenck et al, 2003) in the VBB region to aid in the identification of transgenic events carrying integrated VBB sequences (Okwuonu et al, 2015). DsRed is proven to be a superior fluorescent protein for monitoring gene expression in chlorophyll containing tissues (Zhang et al, 2015).…”