Efficient Plastid Transformation in Arabidopsis

Abstract

Plastid transformation is routine in tobacco (<i>Nicotiana tabacum</i>) but 100-fold less frequent in Arabidopsis (<i>Arabidopsis thaliana</i>), preventing its use in plastid biology. A recent study revealed that null mutations in <i>ACC2</i>, encoding a plastid-targeted acetyl-coenzyme A carboxylase, cause hypersensitivity to spectinomycin. We hypothesized that plastid transformation efficiency should increase in the <i>acc2</i> background, because when ACC2 is absent, fatty acid biosynthesis becomes dependent on translation of the plastid-encoded ACC β-carboxylase subunit. We bombarded <i>ACC2</i>-defective Arabidopsis leaves with a vector carrying a selectable spectinomycin resistance (<i>aadA</i>) gene and <i>gfp</i>, encoding the green fluorescence protein GFP. Spectinomycin-resistant clones were identified as green cell clusters on a spectinomycin medium. Plastid transformation was confirmed by GFP accumulation from the second open reading frame of a polycistronic messenger RNA, which would not be translated in the cytoplasm. We obtained one to two plastid transformation events per bombarded sample in spectinomycin-hypersensitive Slavice and Columbia <i>acc2</i> knockout backgrounds, an approximately 100-fold enhanced plastid transformation frequency. Slavice and Columbia are accessions in which plant regeneration is uncharacterized or difficult to obtain. A practical system for Arabidopsis plastid transformation will be obtained by creating an <i>ACC2</i> null background in a regenerable Arabidopsis accession. The recognition that the duplicated ACCase in Arabidopsis is an impediment to plastid transformation provides a rational template to implement plastid transformation in related recalcitrant crops.