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 ER-targeted 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 greenhouse-grown 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 GFP-expressing 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 co-expression studies.