Monitoring Adventitious Presence of Transgenes in Cotton Collections from Genebank and Experimental Plots: Ensuring GM-Free Conservation and Cultivation of Genetic Resources

Abstract

Bt cotton is the commercialized genetically modified (GM) crop in India with four approved GM events, namely MON531 (Bollgard® I), MON15985 (Bollgard® II), GFM and Event 1. Several GM events of cotton with insect-resistant or herbicide-tolerant GM traits were under field trials or imported for research purposes. With the increase in adoption of Bt cotton, unintentional introgression of transgenes in the conserved diversity-rich germplasm and in the field samples needs to be monitored in a systematic manner employing efficient GM diagnostics. Adventitious presence of transgenes was checked in 100 ex situ accessions of cotton being conserved in the National Genebank. These accessions belonging to Gossypium hirsutum, G. barbadense, G. arboreum, G. hirsutum × G. barbadense, represented seven Bt cotton growing states. Pentaplex PCR assay covering screening for over 90% of globally approved GM cotton events was employed, simultaneously targeting Cauliflower Mosaic Virus 35S promoter (P-35S), Agrobacterium tumefaciens nos terminator (T-nos), neomycin phosphotransferase (nptII) and phosphinothricin-N-acetyltransferase (pat) marker genes, and endogenous stearoyl acyl carrier protein desaturase (Sad1) gene as an internal control. Real-time PCR assays targeting P-35S and T-nos, with the limit of detection up to 0.01%, were employed for further confirmation. A study was also conducted on fifty cotton samples collected from the experimental plots, which were grown at distance of 50 and 10 metres from Bt cotton event MON15985. Adventitious presence of transgenes was checked using multiplex PCR assay targeting cry1Ac, cry2Ab2 and endogenous Sad1 gene to define a strategy for screening for specific GM events. Adventitious presence of transgenes was not detected in any of the tested samples based on the screening tests conducted. This strategy targeting specific GM traits would assist in GM-free cultivation of varieties in the experimental plots. The reported approach with wide GM coverage is reliable for checking adventitious presence of transgenes in genebanks, which could be applied to test conserved germplasm on a regular basis, for ensuring GM-free conservation of genetic resources.