Co-integration, co-expression and inheritance of unlinked minimal transgene expression cassettes in an apomictic turf and forage grass (Paspalum notatum Flugge)

Bahiagrass (Paspalum notatum Flugge) is an important turf and forage grass in the southeastern United States and other subtropical regions. Biolistic co-transfer of two unlinked, minimal, linear transgene expression cassettes (MCs) into the apomictic bahiagrass cv. Argentine was carried out to evaluate co-integration, quantify co-expression and analyze inheritance to apomictic seed progeny. Gold projectiles were coated with minimal unlinked nptII and bar expression cassettes in a 1:2 molar ratio. Complexity of transgene loci correlated with the amount of DNA used during gene transfer. Transgenic plants displayed a simple nptII integration pattern with 1–4 hybridization signals compared to the non-selected bar gene with 2 to more than 5 hybridization signals per transgenic line. Co-expression of unlinked nptII and bar genes occurred in 19 of the 20 co-transformed lines (95% co-expression frequency). Protein quantification revealed that several lines with complex integration patterns displayed a higher transgene expression than lines with simple transgene integration patterns. Several transgenic lines displayed hybridization signals indicative of concatemerization. Concatemers were confirmed following PCR amplification and sequence analysis of transgene loci. The obligate apomictic bahiagrass cv. Argentine produced uniform seed progeny without segregation of simple or complex transgene loci. NPTII- and PAT-ELISA, as well as herbicide application, confirmed stable expression of the nptII and bar gene at levels similar to the primary transformants. These results demonstrate that biolistic transfer of MCs support stable and high level co-expression of transgenes in bahiagrass.