Molecular characterization and field evaluation of glyphosate tolerant sugarcane: a step towards commercial release.

NOGUERA, A.; ENRIQUE, R.; OSTENGO, S.; PERERA, M.F.; RACEDO, J.; COSTILLA, D.; ZOSSI, S.; CUENYA, M.I.; FILIPPONE, M.P.; WELIN, B.; CASTAGNARO, A.P.

Workshop; 8th Molecular Biology Workshop ISSCT (International Society of Sugar Cane Technologist); 2015

ISSCT

Sugarcane commercial variety RA 87-3 was transformed with a genetic construct harbouring the epsps gene from Agrobacterium strain CP4 conferring tolerance to glyphosate and nptII gene for kanamycin-selection of transformed events. Herbicide-tolerance (HT) of transformed sugarcane lines was first evaluated at different concentrations of glyphosate in the greenhouse and later confirmed in field tests. Interestingly, most genetically transformed and HT-lines studied in the greenhouse or in the field showed marked phenotypic changes and/or growth aberrations as compared to the parental line. Therefore, 10 HT-lines, showing phenotypic and growth resemblance to parental variety RA 87-3, were analysed using nine morphologic markers proposed by the International Union for the Protection of New Varieties of Plants (UPOV) and 339 molecular markers (TRAP) to select for good commercial candidates to be further evaluated. Out of the 10 initially selected HT-lines, six showed only minor morphologic and genetic variations and were later field-tested over two vegetative crop cycles (plant-cane and first ratoon) at two production areas, a prerequisite for commercial release of transgenic sugarcane in Argentina. The six field-tested HT-lines were found to be almost indistinguishable when comparing agronomic and industrial characteristics, anti-nutrient content (dhurrin) and chemical composition. In addition, stable heritance of the CP4 epsps gene expression and glyphosate-tolerance throughout different clonal generations were confirmed by RT-qPCR and Southern blot. Taking into account all results, one out of the six lines tested was selected for commercial release and flanking sequences to multiple inserts in the genome of the selected line were determined using Targeted Sequence Enrichment and NGS (Next Generation sequencing) technologies. Our study confirms the utility of genetic transformation as a complementary tool to classical breeding selection in sugarcane and highlights the usefulness of UPOV traits together with molecular markers for early selection of transgenic events that closely resemble their parental genotype.