Improvement of Agrobacterium-mediated transformation frequency in multiple modern elite commercial maize (Zea mays L.) inbreds by media modifications

The current study describes a robust, high-frequency Agrobacterium-mediated transformation protocol suitable for multiple recalcitrant modern elite commercial maize inbreds employing media modifications with glucose, cupric sulfate and a cytokinin, 6-benzylaminopurine (BAP). An optimal combination of these three key elements in the co-cultivation, resting, and selection media resulted in 4- to 14-fold improvements in transformation frequencies at the T0 plant level of 9.7, 31.9, 9.6 and 10.0 % for PH4CN, PH12BN, PHW0V and PH17R8, respectively. Transformation frequency in PH1CP1 was also improved at the T0 tissue level from 2.5 to 8.3 %. The addition of cupric sulfate and BAP in the co-cultivation medium improved transformation frequency in all inbreds except PH4CN. The use of cupric sulfate and BAP in combination with additional glucose in the selection medium was especially important, significantly improving the transformation frequency in 3 (PH4CN, PHW0V and PH1CP1) out of 5 inbreds by increasing the proliferation of high quality regenerable tissue. It was observed that the amount/ratio of these three components needed to be optimized for each inbred. The results in this study can be applied to optimize the tissue culture response and improve transformation frequency in other recalcitrant elite commercial maize inbreds.