Agrobacterium-mediated transformation of sugarcane using GFP as a screenable marker

Abstract

The use of the green fluorescent protein (GFP) and in vivo screening for transgenic cells has enabled the first visual selection of transgenic sugarcane (Saccharum L. hybrid) transformed by Agrobacterium-mediated techniques. Selection of GFP-positive embryogenic callus transformed using Agrobacterium tumefaciensstrain AGL0 was performed by fluorescence microscopy for 6 weeks after transformation. The use of GFP as a screenable marker aided the rapid segregation of individual transformation events and facilitated the initial visual selection of transgenic sugarcane callus without antibiotics, herbicides or an assay. The binary vector used in transformation was pTO134, which contains a synthetic gfp gene (sgfpS65T) and the bialaphos resistance gene (bar) both controlled by CaMV 35S promoters. The appearance of GFP-expressing cells observed on embryogenic callus was 5.3%. Subsequently, GFP-positive calli grew in the presence of a level of the herbicide bialaphos, that was toxic to untransformed calli. GFP-positive shoots were regenerated and one sugarcane plant expressed GFP in the initial stages. Molecular analysis confirmed the presence of the sgfpS65T coding region in the genome of regenerated plants. Visual screening for Agrobacterium-mediated transformation events using GFP was an efficient technique, which may be applied for the transformation of other plants.