SV Joshi1,2, T Makhetha1,2 and M Ghai2
1South African Sugarcane Research Institute, 170 Flanders Drive, Mount Edgecombe, 4300, South Africa; shailesh.joshi@sugar.org.za
2School of Life Sciences, College of Agriculture, Engineering and Science, University of KwaZulu-Natal, Durban 4000, South Africa
The South African Sugarcane Research Institute is developing genetically modified (GM) sugarcane with enhanced resistance to Eldana saccharina by introducing Cry1Ab and Cry2Ab genes. Two genotypes, T10K and TN71, were selected as suitable candidates for transformation, representing South African sugarcane industry’s rainfed and irrigated regions, respectively. Transformed lines were initially screened with lateral flow strips and endpoint polymerase chain reaction (PCR) for the presence of transgenes. This was followed by real-time quantitative PCR (qRT-PCR) and chamber-based digital PCR (cdPCR) to estimate the transgene copy number and expression. Accurate transgene/s copy number measurement is critical for stable resistance expression and to avoid chromosomal effects and gene silencing. GM lines meant for commercial release ideally should have only one or two copies of these genes. Although Southern Blotting is currently regarded as a gold standard method for estimating copy number in various GM crops, it is time-consuming, labour-intensive and expensive. The effectiveness of qRT-PCR versus cdPCR was evaluated for the measurement of gene copy numbers in transformed sugarcane lines. There was variability in copy numbers, with cdPCR offering more efficiency and convenience along with its potential to be more accurate when compared with qRT-PCR. The relationships among transgene copy number, transcript abundance, and transgene protein expression were also determined. cdPCR is recommended for precise measurement of transgene copy numbers in GM sugarcane, as it proved to be more robust, effectual and reproducible. This is the first comparative analysis of the current technologies used to measure ene copy number in transgenic sugarcane. The outcome will allow breeders to accurately correlate eldana resistance levels with copy number and possible collateral effects on yield and sucrose content of the transgenic lines. The cdPCR technology will also enable precise estimation of the inheritance of the transgenes in the progenies.