Enhancing Agrobacterium tumefaciens -mediated transformation efficiency of perennial ryegrass and rice using heat and high maltose treatments during bacterial infection

Perennial ryegrass is one of the most widely cultivated grasses in temperate regions. However, it is recalcitrant for in vitro manipulation. In this study, various parameters affecting Agrobacterium tumefaciens -mediated infection were tested to optimize transformation efficiency in perennial ryegrass. The effects of heat shock and maltose concentration during Agrobacterium infection were evaluated along with variations in callus induction medium, bacterial infection media and callus age. Our results suggest that Agrobacterium infection at 42 °C for 3 min and co-cultivation of Agrobacterium -infected callus on a high maltose medium (6 %) significantly enhances the transformation efficiency in perennial ryegrass. The most optimal conditions proved to be use of four-month-old embryogenic callus induced on a modified N6 medium, infected with Agrobacterium grown on a modified Murashige and Skoog (MSM) medium, and a 42 °C heat shock treatment followed by the co-cultivation of the Agrobacterium and the callus on medium containing 6 % maltose (instead of 3 %). Using this optimized protocol, we were able to increase the transformation efficiencies for regenerated plants from approximately 1 % to over 20 %. Significant improvement in rice stable transformation efficiency was also observed when the optimized conditions were applied to this important cereal, indicating the method described here may apply to other monocots as well.