Over the past 15 years, recombinant DNA and in vitro culture technologies have been used in concert at the South African Sugarcane Research Institute (SASRI) to genetically engineer sugarcane. The purpose of such research has been two-fold, viz. to establish proof-of-principle regarding the delivery of novel input and resistance traits to sugarcane and to investigate the genetic basis of sucrose accumulation. Underpinning these goals has been the in-house development of essential genetic resources, including the isolation of appropriate transgenes and gene promoter elements, and the optimisation of transformation and tissue culture technologies. With regard to the latter, emphasis has been placed on the development of tissue culture strategies that minimise the potential for somaclonal variation, while maximising the efficiency of germplasm transformation, selection, regeneration and acclimation. The isolation of promoters to drive high-level and targeted transgene expression, as well as the identification of DNA sequences with the potential to deliver the desired phenotype, have been strongly driven by advances in gene discovery and expression technologies. Early approaches relied on the analysis of Expressed Sequence Tag libraries constructed from sugarcane tissues at different developmental stages or challenged with selected abiotic and biotic stress agents. Subsequent improvements in DNA subtractive hybridisation technologies (e.g. suppression subtractive hybridisation) and gene expression analysis (e.g. Affymetrix ® Sugarcane Genome GeneChip) provided new and powerful means for gene and promoter discovery. Reviewed here are the advances in in vitro culture, gene discovery and transgene expression research, which have formed the foundation of the genetic engineering program at SASRI. Also discussed are the opportunities and challenges in these areas of research that are presented by the availability of ultra-high throughput DNA sequencing (e.g. the 454 Sequencing System) for transcriptome analysis and technologies for rapid and accurate profiling of the sugarcane metabolomes.