Optimization of Fusion Conditions in the Polyethylene Glycol and the Electric Stimulation Methods for the Protoplasts of Porphyra

Abstract

Fusion of isolated protoplasts (using bacterial crude enzyme following 2% protease treat-ment) from the thalli of Porphyra yezoensis and P. pseudolinearis by the polyethylene glycol (PEG) and the electric stimulation methods is described. In the PEG method, both PEG solu-tion and PEG powder were tested in combination with different dilution media and compared for their fusion rates. The fusion rate of PEG solution and the powder methods did not differ (about 10%). However the electrofusion method yielded higher fusion rates than the PEG method. Optimum fusions were obtained in the protoplasts aligned in an AC field of 40V at 1MHz for 20s and subsequent fusion by a DC pulse of 300V for 40μs duration. Supplementation of Ca²⁺ and Mg²⁺ to the fusion buffer enhanced the fusion rates up to 32.6% (24.3% binary and trinary fusion products) under the same electrical conditions. Protoplasts isolated by bacterial enzyme had higher fusion frequencies than those isolated by using enzymes of top shell and abalone. All the three enzymes showed activity for porphyranase, β-1, 3-xylanase and β-1, 4-mannanase. However enzymes of top shell and abalone had lower activity for porphyranase and β-1, 3-xylanase than the bacterial enzyme.