Production of Transgenic Italian Ryegrass (Lolium multiflorum Lam.) via Microprojectile Bombardment of Embryogenic Calli.

Abstract

We established a microprojectile-bombardment-mediated transformation system in embryogenic calli of Italian ryegrass (Lolium multiflorum Lam.). Bombardment conditions with a PDS-1000/He helium-driven biolistic device were optimized by transient expression assays of a chimeric β-glucuronidase (GUS) gene construct. The highest number of transient GUS expression events was observed with 24 h of high osmotic pretreatment and a bombardment pressure of 7584 kPa. Diploid and tetraploid Italian ryegrass calli were transformed under optimal conditions with two plasmids harboring the gene for antisense expression of cinnamyl alcohol dehydrogenase (CAD) or hygromycin phosphotransferase (HPT). Transformed calli were obtained via microprojectile bombardment followed by selection of transformants on media containing hygromycin at 50mg l-1 for 1 week and then 100mg l-1 for 5 weeks. A total of 33 transgenic plants were produced. The transformation frequencies in cultivars Waseaoba (diploid) and Meritra (tetraploid) were 3.8% and 2.3%, respectively. The total cointegration frequencies of the HPT and antiCAD genes (antisense orientation of CAD gene) were 55.6% in Waseaoba and 73.3% in Meritra. The expression of the antiCAD gene in the co transformed plants was confirmed by RT-PCR.