Isopentenyl Transferase ( IPT ) Gene Transfer to Perennial Ryegrass Through Sonication-Assisted Agrobacterium -Mediated Transformation (SAAT), Vacuum and Heat Treatment

The successful introduction of isopentenyl transferase ( IPT ) gene into perennial ryegrass, cultivars Numan and Grassland using Agrobacterium tumefaciens via three explants (callus, seed and meristem tip) under three individual experiment was evaluated. In the first experiment, the calli were inoculated with LBA4404 Agrobacterium strain under vacuum, heat and in combination of both at 42 °C for 5 min followed by vacuum treatment (390 mm Hg pressure) for 15 min. Sonication-assisted Agrobacterium -mediated transformation (SAAT) was applied for seed and meristem tip transformation of perennial ryegrass for the first time. Results showed positive effects of heat treatment on transformation efficiency during Agro -infection in both cultivars. However, heat shock treatment was more effective in ʻGrasslandʼ than ʻNumanʼ (14.2% vs 9.2%). In addition, high transformation efficiency of about 46.65% and 29.15% was observed using meristem tip explants of ʻGrasslandʼ and ʻNumanʼ based on IPT and RD29A positive PCR results, respectively. Seed transformation efficiency in ʻGrasslandʼ and ʻNumanʼ under SAAT method reached to 37.5% and 16.65%, respectively. Results of these experiments revealed that LBA4404 strain was more efficient than GV3101 in transformation of both perennial ryegrass cultivars. The DNA-blot analysis confirmed that a single T-DNA copy of the IPT gene was integrated into the genomic DNA of the positive transgenic T0 plants which obtained from callus and meristem tip explants of ʻGrasslandʼ after heat and SAAT treatment, respectively. Because monocots are not the host of Agrobacterium tumefaciens , this novel protocol can be used in further experiments on genetic transformation of perennial ryegrass cultivars.