M E N P 1 2 3 4 5 6 7 8 9 10 11 12 M E N P 1 2 3 4 5 6 7 8 9 10 11 12 Fig. 4 PCR detection of transgenes in putative transgenic sorghum lines. a PCR for the nptII gene and b PCR for the gfp gene. Lines from left to right: M 1 kb DNA Ladder (NEW ENGLAND BioLabs). E empty (no template), N non-transgenic Tx430, P plasmid, 1–12 12 samples of putative transgenic lines M N P 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Fig. 5 Southern blot hybridization of 14 putative transgenic lines with nptII probe. Lines from left to right: M DNA ladder. N NC, P equivalent single copy control (pUKN plasmid), 1–14 individual transgenic lines Transgene inheritance assay The gfp gene expression in T1 seedlings was observed under the ﬂuorescence microscope in all tested lines (Fig. 3d). The seedlings with the gfp gene expression were uniformly ﬂuorescent green. However, segregants of the gfp gene was also observed and appeared yellow. Similarly, the expression of the nptII gene in T1 seedlings was conﬁrmed by their survival under G418 selection. T1 segregants of the nptII gene and non-transgenic Tx430 seedlings were necrotic in G418 solution after 1 week 123 --1.0 kb --0.5 kb --1.0 kb --0.5 kb 1004 6kb 3kb a b Plant Cell Rep (2012) 31:999–1007 (Fig. 7a). T1 seedlings with the nptII gene expression were healthy and grew vigorously (Fig. 7b). Discussion Sorghum transformation has been widely considered as challenging since the ﬁrst transgenic sorghum was reported in 1993 (Casas et al. 1993). Here, we report the highest recorded sorghum transformation efﬁciency to date of 20.7%. This result is more than 70 times higher than the ﬁrst successful transformation rate of 0.286% reported via microprojectile transformation, and more than 25 times higher than the biolistic sorghum transformation efﬁcien- cies cited by Grootboom et al. (2010) (0.11–0.77%) and Raghuwanshi and Birch (2010) (0.09%). We have more than doubled the best published frequency of Agrobacte- rium-mediated transformation of up to 8.3% (Gurel et al. 2009). The enhancement of sorghum transformation efﬁ- ciency can be largely attributed to three crucial factors: (1) tissue culture system; (2) DNA delivery system; and (3) selection strategy. The tissue culture system plays a fundamental role in the success of sorghum microprojectile transformation and the Agrobacterium-mediated transformation system. There are many factors which directly inﬂuence the effectiveness of the tissue culture system, from the method of initiating explants to the procedure of planting putative transgenic plantlets in containment glasshouses. Genotype selection is of paramount importance to obtain an efﬁcient tissue cul- ture system. For example, P898012, C401 and Tx430 have been widely studied (Gao et al. 2005b; Sato et al. 2004). In our laboratory, we also routinely transform genotype 296B and SA281, although these do not allow for such a high efﬁciency as Tx430 (data not shown). Choosing the appropriate explant is also important to constantly produce reliable embryogenic callus. IEs have been shown to be the most successful and productive explants for sorghum tissue culture (Elkonin and Pakhomova 2000; Grootboom et al. 2010; Gurel et al. 2009). Optimization of the callus induction medium, regeneration medium and rooting medium is another approach to improve tissue culture performance. M11 medium, which contains MS medium supplemented with 1 g/L KH2PO4, has been shown to be more effective than MS medium for sorghum tissue culture (Sato et al. 2004; Elkonin and Pakhomova 2000). The addition of L-proline and L-asparagine has improved the production of sorghum embryogenic callus and reduced the incidence of toxic phenolic compounds in medium (Elko- nin et al. 1995). Furthermore, the presence of high levels of copper in CIM and regeneration medium has been shown to enhance the callus induction rate and regeneration fre- quency (Nirwan and Kothari 2003). Based on reports in