Efficient production of transgenic cassava using negative and positive selection

In order to improve the efficiency of cassava (Manihot esculenta Crantz) tr ansformation, two different selection systems were assessed, a positive one based on the use of mannose as the selective agent, and a negative one bas ed on hygromycin resistance encoded by an intron-containing hph gene. Trans genic plants selected on mannose or hygromycin were regenerated for the fir st time from embryogenic suspensions cocultivated with Agrobacterium. After the initial selection using mannose and hygromycin, 82.6% and 100% of the respective developing embryogenic callus lines were transgenic. A system al lowing plant regeneration from only transgenic lines was designed by combin ing chemical selection with histochemical GUS assays. In total, 12 morpholo gically normal transgenic plant lines were produced, five using mannose and seven using hygromycin. The stable integration of the transgenes into the nuclear genome was verified using PCR and Southern analysis. RT-PCR and nor thern analyses confirmed the transgene expression in the regenerated plants . A rooting test on mannose containing medium was developed as an alternati ve to GUS assays in order to eliminate escapes from the positive selection system. Our results show that transgenic cassava plants can be obtained by using either antibiotic resistance genes that are not expressed in the micr o-organisms or an antibiotic-free positive selection system.