ISOLATION, CULTURE AND REGENERATION OF SPHEROPLASTS OF BLUE-GREEN ALGAE

Growing cells of Anabaena cylindrica,A.variabilis and Anacystis nidulans were treated with 0.05% lysozyme (w/v) and 2-5mmol·1-1 EDTA,at 35℃ in hypertonic solution.Within 5-8h,about 70-90% of the cells were converted to osmotically sensitive spheroplasts.The effect of culture methods on the formation rate of spheroplasts was studied.The amount of lipopolysaccharides(LPS) released from outer membrane of cells treated with EDTA only was measured.The photosynthetic activity of the spheroplasts and cells suspended in hypertonic solution decreased obviously.The cells suspending in hypertonic solution lost their nitrogen- fixing activity.The spheroplasts of A.cylindrica plated on the agar feed-layer,embedded in the agar medium (0.8-1.5%) or suspended in the culture drop containing 0.5mg·1-1 BA formed the regenerated algae colonies after 9 days.The spheroplasts of A.nidulans embed- ded in the soft agar (0.8%) formed the regenerated algae colonies.Cell division of spheropla- sts suspended in the drop culture could be found on the 4th day of culture.Lysozyme-resis- tance of regenerated algae colonies was compared with that of the cell colonies developed from the residual cells in the lysozyme-treated solution.Spheroplasts of two species were in- duced to fusion with PEG and high pH/Ca,but the attempt failed.