Plant Cell Rep (2012) 31:999–1007 1003 s 40 o y r bm30 e e tru 20 a m ifm10 o . oN 0 GFP expression in immature embryos 3d 10d 1-20 21-50 51-100 No. 0f GFP foci >100 Fig. 2 Transient gfp gene expression in bombarded Tx430 immature embryos (Fig. 4a). The result of gfp PCR analysis showed that 18 of 25 plants were positive (Fig. 4b). Overall, the co-trans- formation frequency of the gfp and nptII genes was 72%. Southern hybridisation conﬁrmed the integration of the nptII gene into the genome of all T0 plants (Fig. 5). Single copy and multiple copies of nptII gene were found in various plant lines. Among the 25 transgenic lines, 24% had a single nptII gene copy, 28% had two to three copies and 48% had at least four copies. Selection of putative transgenics IEs recovered and grew quickly on CIM in the dark 3 days after bombardment. The growth of IEs was clearly con- strained on selective regeneration medium by the applica- tion of G418. Two weeks after selection, most of the IEs displayed necrotic symptoms due to G418. However, a few IEs started to generate shoots. In 4–8 weeks, the transgenic sectors proliferated and produced healthy shoot selection medium (Fig. 6b). Vigorous roots of putative transgenic plantlets were generated on selective rooting medium (Fig. 6c). As much as 100% of all plantlets transferred to pots in the containment glasshouse environment survived (Fig. 6d). The transformation efﬁciency varied from 18.4 to 25% (mean efﬁciency of 20.7%) in the three indepen- dent experiments (Table 2). Fig. 3 GFP expression under the blue light at the wavelength of 395 nm of the OLYMPUS BX60 ﬂuorescence microscope; a Tx430 immature embryo-derived callus 3 days after bombardment; b root tip of non-transgenic Tx430 plantlet; c root tip of transgenic Tx430 plantlet; d 3-day-old T1 seedling 123