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<p>Abstract</p> <p>Background</p> <p>Green algae of the family Volvocaceae are a model lineage for studying the molecular evolution of multicellularity and cellular differentiation. The volvocine alga <it>Gonium </it>is intermediate in organizational complexity between its unicellular relative, <it>Chlamydomonas</it>, and its multicellular relatives with differentiated cell types, such as <it>Volvox</it>. <it>Gonium pectorale </it>consists of ~16 biflagellate cells arranged in a flat plate. The detailed molecular analysis of any species necessitates its accessibility to genetic manipulation, but, in volvocine algae, transformation procedures have so far only been established for <it>Chlamydomonas reinhardtii </it>and <it>Volvox carteri</it>.</p> <p>Results</p> <p>Stable nuclear transformation of <it>G. pectorale </it>was achieved using a heterologous dominant antibiotic resistance gene, the aminoglycoside 3'-phosphotransferase VIII gene (<it>aphVIII</it>) of <it>Streptomyces rimosus</it>, as a selectable marker. Heterologous 3'- and 5'-untranslated flanking sequences, including promoters, were from <it>Chlamydomonas reinhardtii </it>or from <it>Volvox carteri</it>. After particle gun bombardment of wild type <it>Gonium </it>cells with plasmid-coated gold particles, transformants were recovered. The transformants were able to grow in the presence of the antibiotic paromomycin and produced a detectable level of the AphVIII protein. The plasmids integrated into the genome, and stable integration was verified after propagation for over 1400 colony generations. Co-transformants were recovered with a frequency of ~30–50% when cells were co-bombarded with <it>aphVIII</it>-based selectable marker plasmids along with unselectable plasmids containing heterologous genes. The transcription of the co-transformed, unselectable genes was confirmed. After heterologous expression of the luciferase gene from the marine copepod <it>Gaussia princeps</it>, which was previously engineered to match the codon usage in <it>C. reinhardtii</it>, <it>Gonium </it>transformants show luciferase activity through light emission in bioluminescence assays.</p> <p>Conclusion</p> <p>Flanking sequences that include promoters from <it>C. reinhardtii </it>and from <it>V. carteri </it>work in <it>G. pectorale </it>and allow the functional expression of heterologous genes, such as the selectable marker gene <it>aphVIII </it>of <it>S. rimosus </it>or the co-transformed, codon-optimized <it>G. princeps </it>luciferase gene, which turned out to be a suitable reporter gene in <it>Gonium</it>. The availability of a method for transformation of <it>Gonium </it>makes genetic engineering of this species possible and allows for detailed studies in molecular evolution using the unicellular <it>Chlamydomonas</it>, the 16-celled <it>Gonium</it>, and the multicellular <it>Volvox</it>.</p>