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Effects of cell wall-lytic enzymes on the electrofusion efficiency of protoplasts fromPorphyra yezoensis

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DOI: 10.1016/0044-8486(92)90106-u
  • Biology


Abstract The electrofusion efficiency of protoplasts of the marine alga Porphyra yezoensis U. was investigated for the effects of cell wall-lytic enzymes. Fusion products between wild (brown color) and mutant (green color) type protoplasts and between pairs of protoplasts of wild type were visually identified and used to determine the fusion efficiency. Cell wall digestions were carried out in solutions consisting of two kinds of enzymes. One was derived from the marine bacterium Pseudomonas sp. and the other was from one of the following sources: abalones Natohaliotis discus (AAP), top shells Turbo cornutus (TSAP), sea hares Aplysia kurodai (SHAP) or sea urchins Anthocidaris crassispine (SUAP). Both AAP and TSAP, either one combined with bacterial crude enzymes, showed approximately similar effects on the protoplast fusion. Under these enzyme treatments, a maximum of 15 to 20% of the isolated protoplasts formed hybrids after exposure to electric pulses. However, their fusion activities were quite unstable and the fusion rates declined immediately. Thus, fusion rates were only a few percent even when protoplast isolation reached maximum. In the case of the combined SUAP and bacterial enzymes, optimal fusion rate of protoplasts also was observed at approximately 20% but protoplasts could form hybrids with relatively high rates (more than 10%) for at least 2 h during enzyme treatment. Conversely, little or no fusion product was obtained from the protoplasts prepared with the combined SHAP and bacterial enzyme or with the combination of partially purified enzyme from abalone and bacterial, although protoplast production was effective in these solutions. Consequently, the combination of SUAP and bacterial crude enzyme was the most efficient enzyme system for protoplast electrofusion among all the systems examined. In addition, study of the survival rates of the algal protoplasts suggested that the electrofusion efficiency would be largely influenced by the viability of protoplasts during treatment with the wall-lytic enzymes.

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