Robust Mutants Isolated through Heavy-Ion Beam Irradiation and Endurance Screening in the Green Alga Haematococcus pluvialis

Heavy-ion beams have been widely utilized as a novel and effective mutagen for mutation breeding in diverse species, including algae, but a preferred mutant cannot be easily obtained without a suitable large-scale screening method. We devised a unique, convenient, and effective method for screening mutants of Haematococcus pluvialis to isolate a strain resistant to environmental stress with low white fluorescence, i.e., a robust strain. Haematococcus was irradiated with heavy-ion beams of carbon ions, argon ions, and iron ions at various doses, after which approximately 10,000 surviving colonies were inoculated into 96-well plates, cultured for approximately 2 weeks, and then left to dry in a refrigerator for 3–12 months without a lid. In these unattended 96-well plates, cells in approximately one-third of the wells died and became white, and the remaining wells were approximately evenly split between red and green. The robustness of wild-type and mutant strains isolated from red and green wells was compared under severe environmental-stress conditions (125 µmol photons m−2 s−1, continuous light period, 45 mM sodium acetate). In the wild-type strain, most cells died, and 93.9% of cells emitted white autofluorescence. In contrast, few G4 cells emitted white autofluorescence, indicating a survival rate of 91.8%. Strains with excellent carotenoid production, such as G7 and R1, showed greater robustness compared to wild-type strains.