Analysis of transgene integration and expression in genetically modifies sugarcane lines.

Makhetha, T.*¹, Joshi, S.^1,2, Ghai, M.¹

¹ School of Life Sciences, College of Agriculture, Engineering and Science, University of KwaZulu- Natal, Durban, 4000, South Africa
² South African Sugarcane Research Institute, Mount Edgecombe, Durban, 4300

The South African sugarcane industry has realised the benefits of biotechnology for safeguarding sugarcane productivity and is currently developing genetically modified (GM) sugarcane. The particle bombardment method was used to introduce Bacillus thuringiensis genes Cry1Ab and Cry2Ab. These insecticidal transgenes are targeted at the endemic African sugarcane borer pest (Eldana saccharina), which significantly reduces crop yield and threatens the sugar industry. Analysis of transgene expression is an essential step towards selecting a GM sugarcane line that can be chosen for release as a commercial crop. Expression levels can be used to determine the effectiveness of GM lines in providing the required resistance against E. saccharina. Accurate copy number estimation is another vital step, as multiple transgene integration is a common phenomenon after particle bombardment. Multiple copies greatly influence expression levels and genetic stability in GM lines. Techniques such as Southern and Northern blotting have been commonly used for measuring copy numbers and expression levels, respectively. However, their application is time-consuming and labour-intensive. Moreover, both methods can often provide ambiguous results. To overcome these limitations, the standard real-time quantitative PCR (qPCR) and the newly emerged digital PCR (dPCR) platforms can be used. In the current study, two platforms were used and compared for transgene copy number estimation and gene expression analysis of Cry1Ab and Cry2ab genes in T10K and TN71 sugarcane lines. Results of the study indicated variability in transgene copy numbers and expression levels amongst the GM sugarcane lines. Furthermore, the results also indicate dPCR provides greater accuracy and sensitivity in determining transgene copy number and revealing expression levels. This suggests the suitability and routine use of dPCR in evaluating GM sugarcane lines. The current study reports, for the first time, dPCR nanoplate-based assays for both transgene copy number and expression evaluations in GM sugarcane, a highly genetically complex and polyploid crop species.

Keywords: genetically modified sugarcane, Cry transgenes, transgene copy number, digital PCR, transgene expression