Isolation and cell wall regeneration of protoplasts from Posidonia oceanica and Cymodocea nodosa

A method for the isolation of protoplasts from the seagrasses Posidonia oceanica and Cymodocea nodosa is described. Isolation of protoplasts was achieved using a combination of cellulase Onozuka R-10, hemicellulase and pectinase. Purification was carried out by Ficoll gradient centrifugation. A yield of 1.87 (+/- 0.16 SD) x 10(6) protoplasts per gram of fresh tissue was obtained from mesophyll cells of P. oceanica. The viability of isolated protoplasts was 82.5% (+/- 10.6) as confirmed by fluorescein diacetate staining. A high percentage of protoplasts of Fl oceanica (61.5 +/- 14.8%) regenerated the cell wall within 7 days as confirmed by staining with calcofluor white, but only a few protoplasts were able to divide. Callus-like structures were noticed after 20-30 days in culture. A lower yield of protoplasts, 6.9 (+/- 3.8) x 10(5) protoplasts per gram of fresh tissue, was obtained from mesophyll of C. nodosa. Viability of these protoplasts was 67.9% (+/- 12.6) after isolation. Some possible applications of the method are discussed. (C) 2001 Elsevier Science B.V. Ah rights reserved.