Stable chloroplast transformation in Chlamydomonas reinhardtii using microprojectile bombardment

The chloroplasts ofChlamydomonas reinhardtii were transformed using a vector (paadAGUS4.1) that contained a spectinomycin-resistance gene (aadA) as a selectable gene, and bacterialuidA (GUS) as a reporter gene, and pea 4.1 kb D-loop containing sequence. The vector was introduced into the alga through particle gun bombardment. The transformed colonies were screened for the presence of foreign genes by Southern hybridization using GUS,aadA and 4.1 pea Ori probes. Expression ofaadA and GUS genes was detected in all colonies that were grown on spectinomycin. A detailed restriction analysis followed by southern hybridization of total genomic DNA using pea 4.1 kb D-loop as probe indicated that the D-loop sequence can serve in site-specific integration of foreign DNA due to high homology. Restriction analysis of different colonies showed that the foreign DNA was probably present in a mixture population of autonomous segment and integrated in the native chloroplast genome.