Efficient Approaches for Nuclear Transgene Stacking in the Unicellular Green Microalga Chlamydomonas Reinhardtii

Genetic crossing is routinely applied to alter multigenic traits in the unicellular model green microalga Chlamydomonas reinhardtii (Chlamydomonas). However, efficient antibiotic resistance-based screening for the desired progenies is impossible through the conventional approach when two parental strains are harboring the same antibiotic marker. In this study, we describe a simplified and efficient genetic crossing method for transgene stacking and progeny testing with the desired traits in Chlamydomonas strains harboring the same antibiotic marker. Our hereby-described approach allowed for the successful generation of dual transgene-expressing strains after crossing different mating type transformants harboring the same marker gene. Moreover, we employed a polyethylene glycol (PEG)-mediated protoplast fusion approach as an alternative method to perform transgene stacking in strains harboring the antibiotic markers but with low mating efficiency or mating inability. Using our optimized method, we successfully generated cells expressing several transgenes regardless of their mating efficiency. These methods could be potentially applicable and useful to other unicellular microalgal species.