An improved regeneration protocol suitable for transformation of sorghum was developed. The improvements focused on limiting the production of phenolic compounds and the use of suitable culture vessels for each developmental stage in plant regeneration from immature embryo derived calli. The addition of activated charcoal in the callus induction medium reduced the production of black pigments, however it also inhibited the callus formation on immature embryo explants. Cold pre-treatment of the immature seeds from which embryo explants were excised had a positive effect on both explant survival and callus formation. A one-day 48C treatment of immature seeds significantly improved the callus formation from immature embryos and reduced the need for frequent subculture. Petri dishes with ventilation were suitable for the callus induction phase, but not for plant regeneration. Regeneration of plants could be improved by using disposal plastic boxes (250 ml volume) instead of Petri dishes. Agrobacterium-mediated transformation using the improved regeneration protocol and the hygromycin phosphotransferase gene as selectable marker resulted in the recovery of 15 transgenic plants from 300 initial immature embryos (5% efficiency). The transgenic nature of the obtained plants was demonstrated by Southern hybridisation and progeny analysis. The transgenes were inherited in a Mendelian fashion and were integrated at a single locus in the majority of the analysed lines.

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