PEG-Induced Cell Fusion Of Haematococccus Pluvialis And Screening Of Variants

Haematococcus pluvialis, an fresh-water green unicellular algae, is presumed to be an important algae for its secretion of an valuable red pigment---astaxanthin. Astaxanthin is favourable colorant and has function in anti-oxidation, acquisition of free radical, anti-canceration, anti-ultraviolet radiation as well as improvement of immunity. It is wildly needed in the fields of feed, food and comics. But so far the output is very low, currently, intensive cultivation of astaxanthin is extended both at home and abroad. However, problems such as low growth rate and contaminations are still existed; therefore, breeding heavy producing algae is the key point in cultivation.In this study, different concentrations of PEG was used as mutagen to induce cell fusion, for crossbreeding variation, cells were also treated in different concentrations of Ca2+ and dimethyl sulfoxide dimethyl sulphoxide (DMSO)with different treated time. Orthogonal test was designed, volume fraction of PEG was detected, and the influence on cell fusion rates of these above factors was valued.The results showed that highest fusion rate was achieved when cells were touched with the shaking-beating method, and were treated by 55% PEG,25mg/1000ml Ca2+,10% DMSO with 10 min at 25℃. A4B2C3D2 was the optimal combination. Meanwhile, when cells were touched with the combinated methods of shaking-beating and extruding by centrifugal force, the highest fusion rate was achieved when cells were treated by 50% PEG, 25mg/1000ml Ca2+,5%DMSO at 25℃with 10min. Ac3Bc2Cc2Dc2 was the optimal combination.Cells were screened by methods of solid medium and centrifugation, divided number was calculated by using microscope, diameter was measured, cellular volvocine algae with cell numbers more than 8 and diameter more than 25 were selected, results showed that 20 cell strains with mast cells and fast division speed were screened. It was concluded that the combination method with centrifugation and solid plate culture medium was the optimal operation.The 20 selected Haematococcus pluvialis strains were cultured in MVA fluid medium. Growth rates were valued when strains were exposed to 2000Lux, then they were exposed to 5000-6000Lux, astaxanthin accumulation and output were detected. Ultimately, strains of P5, P13 and P20 were selected by considering the growth rate and the output. Compared to the original strains, these parameters were increased by 30.23%,19.86%,19.16% and 72.12%,73.96%,63.89%, respectively.After strains of P5, P13 and P20 were cultured for four to five generations, the growth rate and the output of astaxanthin were continually detected, results showed that these traits were sustainable, therefore, it was suggested that the screened variant was heavy producing.