Development of marker-free transgenic sorghum ... - Plant Sciences

100 Plant Cell Tiss Organ Cult (2009) 99:97–108Fig. 2 Sorghum transformation, plantlet regeneration, GUS assayand leaf-painting analysis of transgenic events. a Embryos wereco-cultivated with Agrobacterium on TM for 3–5 days; b Resting onRM for additional 4 days; c Callus induction under selection pressure,herbicide-tolerance calli developed from some embryos on CM;d Shoot formation on SM; e After 2–3 weeks of being transferredonto ZM, shoots began rooting; f and g Putative transgenic plants (youngand adult) were in greenhouse; h GUS assay of embryogenic calli;i leaf painting showing susceptible response of wild-type leaves (lefttwo leaf strips) and resistance of transgenic leaves (right two strips)The embryos were transferred onto resting medium(RM) for another 4 days at 28°C in the dark (Fig. 2b) andthen transferred weekly onto freshly made callus inductionmedium (CM) with PPT 2.5 mg l -1 , incubated at 28°C for4–8 weeks (Fig. 2c). Herbicide-tolerant calli can be maintainedon CM, but untransformed calli eventually die. Oncesomatic embryogenic cells developed from transformedcalli, they were transferred onto shooting medium (SM)(Fig. 2d). Shoots (2–5 cm long) were separated and transferredinto plastic boxes (898910 cm) containing rootingmedium (ZM), and maintained at 25°C under a photoperiodof 16 h light and 8 h dark; the shoots later developed into8–10 cm tall plantlets with healthy roots (Fig. 2e). Theywere then transferred into pots containing Promix soil mixedwith Osmocote (18-6-12) in the greenhouse (Fig. 2f and g).GUS assay and leaf painting using libertyÒTo verify the expression of GUS gene, calli and plant tissueswere assayed with histochemical X-Gluc staining at 37°Cfor 24 h (Fig. 2h; Jefferson et al. 1987). The X-Gluc solutionfor staining target tissues contained: 0.1 M NaPO 4 ,pH7.0; 1.0 mM K 3 [Fe(CN) 6 ]; 10 mM EDTA; 2 mM X-Gluc;0.1% (v/v) Triton-100; 20% (v/v) methanol anhydrate.123