Analysis of transgene integration and expression following biolistic transfer of different quantities of minimal expression cassette into sugarcane (Saccharum spp. hybrids)

Abstract

Sugarcane (Saccharum spp. hybrids) is an interspecific hybrid with a highly polyploid and frequently aneuploid genome. This C4 grass accounts for nearly 70% of the global sugar production and more recently has become an important biofuel feedstock. Biolistic gene transfer of plasmid DNA is the most frequently used approach for genetic transformation of sugarcane. Minimal expression cassettes lacking vector backbone sequences (MC) have been reported to support simple transgene integration in other species. In this study, we introduced a MC of nptII into embryogenic callus derived from immature leaf whorl cross-sections by biolistic gene transfer. The precipitation equivalents of 12.5, 25 or 50 ng of the nptII MC were delivered per shot to the target tissue with 1.0 μm gold particles. A total of 203 independent putative transgenic plants were regenerated following 80 bombardments and selection on geneticin or paromomycin containing media and 176 transgenic lines were confirmed with PCR. Twenty independent transgenic lines were selected for Southern blot analysis and expression analysis by NPTII ELISA from each of the three treatments. Genomic DNA from transgenic sugarcane plants displayed two to 13 nptII hybridization signals on Southern blots. There was a trend toward reduced transgene integration complexity and reduced transgene expression levels when lower (12.5 ng) MC was used per shot. These results demonstrate that backbone free MCs can be efficiently integrated and expressed in sugarcane.