High-efficiency Agrobacterium-mediated transformation of Lotus corniculatus L. using phosphomannose isomerase positive selection

摘要

A method for an Agrobacterium-mediated transformation with phosphomannose isomerase-based selection system was investigated for cotyledons explants of Lotus corniculatus L. cultivar Leo. The effects of mannose on shoot formation and rooting was evaluated to choose appropriate selection pressure for Lotus corniculatus L. transformation. Complete inhibition in Murashige and Skoog medium supplemented with 20 g/L mannose in the presence of 20 g/L sucrose was observed, indicating that Lotus corniculatus L. is incapable of utilizing mannose as a carbon source. The mannose-resistant shoots from individual transformed lines selected in early developmental stage were identified by PCR analysis, resulting in a transformation frequency of 16.2 %. Six randomly chosen PCR-positive plantlets were further confirmed by Southern-blotting and RT-PCR analysis to demonstrate integration of the phosphomannose isomerase (PMI) gene. Chlorophenol red assays were performed to determine PMI enzyme activity. These results demonstrate that the PMI-based selection is a highly efficient system for Lotus corniculatus L. transformation without requiring the use of conventional antibiotics or herbicide marker genes.