A simple and reliable Agrobacterium -mediated transformation method was developed for switchgrass. Using this method, many transgenic plants carrying multiple genes-of-interest could be produced without untransformed escape. Switchgrass (Panicum virgatum L.) is a promising biomass crop for bioenergy. To obtain transgenic switchgrass plants carrying a multi-gene trait in a simple manner, an Agrobacterium-mediated transformation method was established by constructing a Gateway-based binary vector, optimizing transformation conditions and developing a novel selection method. A MultiRound Gateway-compatible destination binary vector carrying the bar selectable marker gene, pHKGB110, was constructed to introduce multiple genes of interest in a single transformation. Two reporter gene expression cassettes, GUSPlus and gfp, were constructed independently on two entry vectors and then introduced into a single T-DNA region of pHKGB110 via sequential LR reactions. Agrobacterium tumefaciens EHA101 carrying the resultant binary vector pHKGB112 and caryopsis-derived compact embryogenic calli were used for transformation experiments. Prolonged cocultivation for 7 days followed by cultivation on media containing meropenem improved transformation efficiency without overgrowth of Agrobacterium, which was, however, not inhibited by cefotaxime or Timentin. In addition, untransformed escape shoots were completely eliminated during the rooting stage by direct dipping the putatively transformed shoots into the herbicide Basta solution for a few seconds, designated as the 'herbicide dipping method'. It was also demonstrated that more than 90 % of the bar-positive transformants carried both reporters delivered from pHKGB112. This simple and reliable transformation method, which incorporates a new selection technique and the use of a MultiRound Gateway-based binary vector, would be suitable for producing a large number of transgenic lines carrying multiple genes.

Switchgrass (Panicum virgatum L.) is an important bioenergy crop. A reliable and efficient transformation method is required to assist with molecular breeding of this crop. In this study, we established a simple and efficient Agrobacterium-mediated transformation method for caryopsis-derived Type I callus by optimizing the cocultivation (7 days at 22°C on medium supplemented with 10 g l −1 glucose and 100 µM acetosyringone) and preculture (2 weeks on medium supplemented with 5 g l −1 casamino acids after cell straining) conditions without the need for time-consuming treatments before and after cocultivation. The present transformation method was successfully applied to different genotypes of switchgrass including a recalcitrant lowland cultivar. The transformation efficiencies of callus lines of the lowland cultivars ' Alamo' and 'Kanlow' were 12.5-59% and 6.3-20%, respectively. An upland cultivar 'Trailblazer' formed Type I calli and one of three tested callus lines produced transgenic plants at relatively high efficiency (7.5%). In contrast, Type I callus formation was unsuccessful for two other upland cultivars, 'Blackwell' and 'Cave-in-Rock. ' This simple and efficient transformation method is suitable for routine and large-scale experiments due to its ready availability of caryopses, prevalence of Type I callus formation, and longevity in the regeneration ability of the callus.