Plant Cell Rep (2012) 31:999–1007 1005 Fig. 6 The development of putative transgenic sorghum plants. a The initiation stage—immature embryo-derived calli ready for micropro- jectile transformation; b putative transgenic shoots on selective (30 mg/L G418) regeneration medium at 6 weeks; c putative trans- genic plantlet on selective (30 mg/L G418) rooting medium at 4 weeks; d putative transgenic plants in containment glasshouse literature, we optimized the usage of additional ingredients including CuSO4, KH2PO4, L-proline, L-asparagine and plant growth regulators including 2,4-D, IAA, IBA, NAA and BAP in callus induction medium, regeneration medium and rooting medium. After optimization of the sorghum tissue culture system for inbred line Tx430, an efﬁcient tissue culture system was established. More than 95% of IEs form callus, and of these more than 70% regenerate shoots with no further loss of in vitro plantlets during the transition from the tissue culture environment to contain- ment glasshouse conditions. Promoters and reporter genes play an important role in optimizing the DNA delivery system. There are many promoters which have been studied for plant transforma- tion, but the strength and suitability of promoters are var- iable and result in different levels of gene expression in target tissue (Kumar et al. 2011; Tadesse et al. 2003; Able et al. 2001). It was reported that the number of GUS foci was signiﬁcantly higher from ubi1 than from actin1 and CaMV 35S (Able et al. 2001). Similarly, this result was further reﬁned utilizing GUS histochemical staining and Table 2 Transformation efﬁciency of three independent experiments with immature embryo (IE)-derived calli of Sorghum bicolor Tx430 Experiment Bombarded IE Transgenic events Transformation efﬁciency (%) 1 2 3 Total 49 36 36 121 9 7 9 25 18.4 19.4 25.0 20.7 enzymatic activity assay, showing the strength of promot- ers in descending order: ubi1 [ act1D [ adh1 [ CaMV 35S (Tadesse et al. 2003). Recently, CaMV 35S promoter activity was reported to be very low in small T0 sorghum transgenic plants (Kumar et al. 2011). The ubi1 promoter has proven to be the most successful promoter in sorghum transformation in recent years (Grootboom et al. 2010; Raghuwanshi and Birch 2010; Gurel et al. 2009). GFP is a highly versatile reporter gene, because the gfp gene expression can be monitored any time in living cells under a ﬂuorescence microscope in a non-destructive manner 123