Marker free transgenic plants: engineering the chloroplast genome without the use of antibiotic selection

Chloroplast genetic engineering offers several advantages over nuclear transformation including high levels of gene expression and gene containment. However, a consequence of placing a transgene in the chloroplast genome is that the antibiotic resistance genes used as selectable markers are highly amplified. Engineering genetically modified (GM) crops without the use of antibiotic resistance genes should eliminate the potential risk of their transfer to the environment or gut microbes. Therefore, the betaine aldehyde dehydrogenase (BADH) gene from spinach was used in this study as a selectable marker. The selection process involves conversion of toxic betaine aldehyde (BA) by the chloroplast BADH enzyme to non-toxic glycine betaine, which also serves as an osmoprotectant. Chloroplast transformation efficiency was 25-fold higher in BA selection than with spectinomycin. In addition, rapid regeneration was obtained. Transgenic shoots appeared within 12 days in 80% of leaf disks (up to 23 shoots per disk) under BA selection compared to 45 days in 15% of disks (1 or 2 shoots per disk) under spectinomycin selection. Southern blots confirmed stable integration of foreign genes into all of the chloroplast genomes (approximately 10,000 copies per cell) resulting in homoplasmy. Transgenic tobacco plants showed 15- to 18-fold higher BADH activity at different developmental stages than untransformed controls. Transgenic plants were morphologically indistinguishable from untransformed plants and the introduced trait was inherited stably in the subsequent generation. This is the first report of genetic engineering of the higher plant chloroplast genome without the use of antibiotic selection. The use of naturally occurring genes in spinach for selection, in addition to gene containment, should ease public concerns regarding GM crops.