TRANSGENIC PERENNIAL RYEGRASS (LOLIUM-PERENNE) PLANTS FROM MICROPROJECTILE BOMBARDMENT OF EMBRYOGENIC SUSPENSION CELLS

Transgenic forage-type perennial ryegrass (Lolium perenne L.) plants have been obtained by microprojectile bombardment of embryogenic suspension cells using a chimeric hygromycin phosphotransferase (hph)gene construct driven by rice Act1 5' regulatory sequences. Parameters for the bombardment of embryogenic suspension cultures with the particle inflow gun were partially optimized using transient expression assays of a chimeric beta-glucuronidase (gusA) gene driven by the CaMV 35S promoter. For the recovery of stably transformed clones, hygromycin selection using liquid and solidified media was tested. Initial selection in liquid culture medium allowed for a higher, compared with continuous plate selection using solid medium, recovery efficiency of transformed hygromycin resistant clones. Plants were regenerated from 23% of the hygromycin resistant calli obtained. The transgenic nature of the regenerated plants was demonstrated by Southern hybridization analysis. Expression of the transgene in transformed adult perennial ryegrass plants was confirmed by Northern analysis and a hygromycin phosphotransferase enzyme assay.