1002 GCGATAGAAGG-30, and GFP-speciﬁc primers: GFP-1: 50-CCGCGGTTACTTGTACAGCTCGTCC-30; GFP-2: 50-CCCGGGATGGTGAGCAAGGGCGAGG-30 (INTE- GRATED DNA TECHNOLOGIES, www.idtdna.com). PCR analyses were performed in 25-lL reaction mixtures, each containing 50 ng of template DNA, 19 PCR buffer, 200 lM of each dNTP, 2.5 mM MgCl2, 0.5 lM primer and 2 U Taq DNA polymerase (NEW ENGLAND BioLabs). The PCR program utilized for the nptII gene consisted of an initial denaturation at 95 C for 5 min, followed by 35 cycles, consisting of 94 C for 30 s, 60 C for 30 s, 72 C for 50 s and a ﬁnal 7-min elongation step at 72 C. Similarly for the gfp gene PCR reactions, the annealing temperature was changed to 70 C for 30 s. PCR products were sepa- rated by gel electrophoresis in 1.0% agarose gels. Southern hybridization analysis Aliquots of *10 lg total genomic DNA of each putative transgenic sample and non-transgenic Tx430 were digested with the restriction enzyme SacI-HFTM (NEW ENGLAND BioLabs) at 37 C overnight. As the positive control, 100 pg of undigested pUKN plasmid was used. The digested DNA and plasmid were then separated on a 0.8% agarose gel for 16 h at 25 V. DNA samples were then transferred to a positively charged nylon membrane (Roche) via blot apparatus overnight. The following day, the membrane was hybridized using the digoxigenin (DIG)-labeled nptII probe at 42 C overnight (probe con- struction was carried out using nptII primers following the PCR conditions described previously). The next day, the membrane was washed, blocked and ﬂuorescent signal detected according to manufacturer instruction (Roche). Transgene inheritance assay Six PCR-conﬁrmed T0 lines containing both nptII and gfp genes were selected for analysis of gene inheritance. Non- transgenic Tx430 seeds were used as a control. For each selected line, 25 seeds were placed onto a petri dish of 25 9 90 mm, lined with two ﬁlter papers (Whatman No. 1). Seeds were then partially covered by sterilized water and allowed to germinate in the dark at 27 ± 1 C for 3 days. The gfp gene expression in the T1 seedlings was monitored utilizing the OLYMPUS BX60 ﬂuorescent microscope. Seedlings were then soaked in sterilized water containing 30 mg/L G418. Plates were placed under light in the tissue culture room for 1 week. Observations of the health and vigor of seedlings for each treatment were recorded, with particular attention paid to detecting any signs of necrosis among the seedlings under G418 selection. 123 Plant Cell Rep (2012) 31:999–1007 Results Kill curve IEs began to display G418 stress symptoms on selective regeneration media within 1 week. IEs on regeneration medium without G148 (G0) grew healthily and showed the beginnings of shoots within 2 weeks and generated vigorous shoots within 4 weeks. On G0 medium, all IEs survived and 14 of 15 IEs produced vigorous shoots (Table 1). In contrast, none of the IEs generated shoots on G30 or G50 media, and the majority were necrotic. After 4 weeks, the results dem- onstrated that G418 had a signiﬁcant impact on callus growth and development in selective media with different G418 concentrations (G0, G10, G20, G30 and G50) (Table 1). Even though two and six IEs produced shoots on G20 and G10 media, respectively, the shoots were shorter and weaker than those on G0 media. The pattern in the kill curve experiment was observed as follows: the higher the G418 concentration in selective regeneration media, the lower the shoot regeneration frequency was evident and the more necrotic IEs were observed (Table 1). In this experi- ment, 30 mg/L G418 in medium was sufﬁcient to prevent IEs from producing shoots and was therefore selected as the optimal concentration for Tx430 transformation. GFP observations Strong gfp gene expression was observed in all bombarded IEs with at least 20 GFP foci. More than 100 GFP foci were spotted in about 80% of IEs (Figs. 2, 3a). When examining root tips of putative transgenic plantlets, the gfp gene expression was shown to be systemically green under the ﬂuorescent microscope (Fig. 3c). No GFP was found in root tips of non-transgenic plantlets and the root tips of these plantlets displayed yellow color under the ﬂuorescent microscope (Fig. 3b). Molecular analysis of putative transgenic (T0) lines PCR results conﬁrmed the presence of the NPTII gene in all transgenic lines and no nptII escapes were observed Table 1 G418 kill curve of Tx430 immature embryos (IEs) under lights in 4 weeks Selective media G418 (mg/L) IE with shoots IE survival G0 G10 G20 G30 G50 0 10 20 30 50 14 6 2 0 0 15 11 8 3 1