1000 2007; Gurel et al. 2009; Able et al. 2001). Recently, up to 8.3% of stable Agrobacterium-mediated transformation efﬁ- ciency has been reported (Gurel et al. 2009), although this was the best single experiment among a number using Agrobacterium-mediated transformation. However, there have been few signiﬁcant advances in microprojectile trans- formation efﬁciency (Grootboom et al. 2010; Raghuwanshi and Birch 2010). Grootboom et al. obtained 27 transgenic plants from 3,499 immature embryos (IEs) of the cultivar P898012 utilizing manA gene, and the transformation efﬁ- ciency at 0.77% was more than twofold higher than the ﬁrst published attempt (0.286%) (Casas et al. 1993). Raghuwanshi and Birch obtained 16 transgenic plants of sweet sorghum from 17046 IEs bombarded. Even though the tissue culture system was optimized, the transformation efﬁciency of sweet sorghum (0.09%) was much lower than the ﬁrst published attempt (0.286%) of grain sorghum (Casas et al. 1993). A highly efﬁcient gene transfer system largely rests on an effective tissue culture system and an optimal DNA delivery system. Important progress has been made in both areas (Kaeppler and Pedersen 1997; Elkonin et al. 1995; Elkonin and Pakhomova 2000; Nirwan and Kothari 2003; Able et al. 1998; Karami et al. 2009; Carvalho et al. 2004; Hill-Ambroz and Weeks 2001; Jeoung et al. 2002; Able et al. 2001), but sorghum transformation efﬁciency remained signiﬁcantly below 10% and was far behind the transformation efﬁciencies of rice 50–90% (Hiei and Komari 2008), maize 50% (Ishida et al. 2007) and barley 14.8% (Ibrahim et al. 2010). An efﬁcient genetic transformation system plays a key role in molecular breeding and understanding the genetic control of plant physiology and development. Sequencing of the sorghum genome was completed in 2009 (Paterson et al. 2009). Genome sequencing and gene discovery have gained momentum in the last decade. Yet, the absence of an efﬁcient sorghum transformation protocol has hampered progress in sorghum genetic engineering and understanding of sorghum gene function. The aim of the present study was to develop a highly efﬁcient transformation system for sorghum. Focusing on an extensively used inbred sorghum line Tx430 (Gurel et al. 2009), we optimized callus induction medium (CIM), regeneration medium and rooting medium to reduce the production of phenolics from tissue and increase the callus induction rate and regeneration frequency. Other transfor- mation variables were also optimized. Materials and methods Plasmids 123 Two plasmids, pUKN and pGEM-ubi-gfp (Dugdale et al. 1998), which contain nptII encoding NPTII and gfp Plant Cell Rep (2012) 31:999–1007 encoding GFP, respectively, were kindly provided by Scott Hermann, Bureau of Sugar Experimental Stations (BSES) Limited. Both genes were driven by the maize ubiquitin1 (ubi1) promoter and terminated with A. tum- efaciens nopaline synthase 30 termination signal (nos) (Fig. 1a, b). Plant materials and embryo isolation Seeds of the inbred line Tx430 (Miller 1984) were planted in the temperature control glasshouse (18–28 C) and some were planted outside in the ﬁeld during the summer. Plants were watered daily and fertilized with Osmocote monthly. Panicles were covered with paper bags before ﬂowering and immature seeds were harvested within 12–15 days after ﬂowering. To sterilize immature seeds, they were soaked in 70% ethanol (v/v) while shaking at 200 rpm for 5 min. Seeds were rinsed once with sterilized water, before being soaked again in the solution containing commercial bleach (White King) 4% (v/v) sodium hypo- chlorite and 0.2% (v/v) surfactant Tween 20 while shaking at 200 rpm for 10 min. Finally, seeds were rinsed ﬁve times with sterilized water. IEs ranging from 1.0 to 2.0 mm in length were isolated onto 90 9 15 mm petri dishes containing CIM with their scutellums facing upward. Cultures were incubated in the dark in a tissue culture room, which was set up at 27 ± 1 C under ﬂuorescent light of approximately 100 lmol s-1 m-2 (16 h/day). Media for tissue culture All media were based on MS medium (Murashige and Skoog 1962) containing 4.44 g/L MS powder with Gam- borg vitamins (PhytoTechnology Laboratories), 30 g/L sucrose and 8 g/L agar. The pH of all media was adjusted to 5.7 before being autoclaved at 121 C for 15 min; 2,4-D was added to media before autoclaving. The other plant growth regulators and copper sulfate (CuSO4) were sterilized with a 0.2-lm syringe ﬁlter and added after autoclaving. The following media were used for microprojectile transformation experiments: Callus induction medium (CIM): MS medium supple- mented with 1 g/L L-proline, 1 g/L L-asparagine, 1 g/L potassium dihydrogenphosphate (KH2PO4), 0.16 mg/L CuSO4 and 1 mg/L 2,4-D. Regeneration medium: MS medium supplemented with 1 mg/L BAP, 1 mg/L IAA and 0.16 mg/L CuSO4. Rooting medium: MS medium supplemented with 1 mg/L NAA, 1 mg/L IAA, 1 mg/L IBA and 0.16 mg/L CuSO4. Selective regeneration medium: regeneration medium supplemented with 30 mg/L geneticin (G418) (disulfate salt solution, SIGMA).