Tools for chloroplast transformation in Chlamydomonas: expression vectors and a new dominant selectable marker

Reverse-genetic studies of chloroplast genes in the green alga Chlamydomona s reinhardtii have been hampered by the paucity of suitable selectable mark ers for chloroplast transformation. We have constructed a series of vectors for the targeted insertion and expression of foreign genes in the Chlamydo monas chloroplast genome. Using these vectors we have developed a novel sel ectable marker based on the bacterial gene aphA-6, which encodes an aminogl ycoside phosphotransferase. The aphA-6,4-6 marker allows direct selection f or transformants on medium containing either kanamycin or amikacin. The mar ker can be used to inactivate or modify specific chloroplast genes, and can be used as a reporter of gene expression. The availability of this marker now makes possible the serial transformation of the chloroplast genome of C hlamydomonas.