Establishment and optimization of PEG-mediated protoplast transformation in the microalga Haematococcus pluvialis

Abstract Genetic manipulation of Haematococcus pluvialis is difficult because the lack a stable and convenient transformation system. The pH124-EGFP-Ble vector containing ble as selective gene EGFP reporter was constructed employed for effective transformation. H. protoplasts were obtained by treating with cellulase macerozeme. Then polyethylene glycol-mediated established incubating protoplast vector. To improve efficiency protoplasts, system optimized in consideration different influencing factors, including zeomycin concentration, growth stage, amount transformed vector, linearization duration low-intensity illumination. integration expression confirmed transformants. Moreover, optimal combination determined to be 5 µg linearized used transform cells log phase, then allowed recover under illumination 6 h. This study represents describes successful development an protocol using which will enable genetic this important algal species.