Professor Farmingdale State College Farmingdale, New York
Body of Abstract: Chloroplast transformation is a powerful tool for plant genetic engineering because plastids exhibit maternal inheritance, high gene expression levels and organization of genes in operons. Chloroplast transformation is routine in tobacco but in the model plant Arabidopsisthaliana, 200 initial attempts at chloroplast transformation yielded only two transplastomic plants that were both sterile. The low transformation efficiency occurred because Arabidopsis is naturally tolerant to spectinomycin, which is used for transformant selection. The two regenerated transplastomic plants were sterile due to the poor regeneration capacity and polyploid nature of the Arabidopsis leaf tissue used for the experiments. A nuclear-encoded acc2 gene, which is duplicated in many Brassicaceae plants, was responsible for the spectinomycin-tolerance. Inactivation of the duplicated acc2 gene resulted in spectinomycin-sensitivity and increased transplastomic event identification 100-fold. In this study, we introduced a steroid-inducible Baby Boom (BBM) gene into the spectinomycin-sensitive Arabidopsisacc2 plant lines to improve their regeneration capacity. Leaves from the Arabidopsisacc2-BBM plant lines were used as source tissue for spectinomycin-selection experiments. Eighteen spectinomycin-resistant calli were obtained from 18 bombardments. Flowering plants regenerated from eight of the calli and viable seed formed from several plants. Molecular characterization of DNA from the regenerated plants determined that spectinomycin resistance was due to spontaneous mutations in the chloroplast encoded rrn16 gene. Coupling of the spectinomycin-sensitivity with the improved plant regeneration system reported here, resulted in reproducible regeneration of fertile spectinomycin-resistant plants from regenerated leaf tissue in Arabidopsis. It is expected that utilization of this protocol will enable chloroplast transformation in Arabidopsis to become routine, which will have a major impact on basic science and applications in biotechnology due to the extensive genomic resources available for the model plant.