Assembly of the subcellular parts of Bryopsis hypnoides Lamouroux into new protoplasts

Abstract

The chloroplasts, mitochondria, and protoplasm devoid of mature chloroplasts (PMC) of Bryopsis hypnoides Lamouroux were isolated by low-speed and sucrose density centrifugation. The PMC aggregated in artificial seawater, and then protoplasts without mature chloroplasts (PtMCs) were formed. Transmission electron microscopy and cytochemical studies indicated that there were mitochondria, nuclei, vesicles, and other small cell organelles in the PtMCs. Scanning electron microscopy showed that there were holes on the surface of 1-h PtMCs and then fewer holes on the surface of 24-h PtMCs, suggesting that a healing process occurred. The plasma membrane was formed over the surface of the PtMCs. However, the cell wall was not regenerated, and the newly formed PtMCs were ruptured and died in 3 days. Light intensity during alga maintenance before use influenced significantly (one-way ANOVA, P < 0.0001) on the number of PtMCs formed; the highest number of PtMCs was formed at 20A mu mol/(m(2) s). When isolated chloroplasts were transferred into seawater, there were only two or three chloroplasts aggregated together. However, isolated mitochondria and the mixed six layers of cell organelles (separated by sucrose density centrifugation) could not aggregate in the artificial seawater. This indicates that the conjunction of cell organelles is important for their aggregation.