Hormone-autonomous cell culture from cotydelonary tissue of the marine plant Cymodocea nodosa

In vitro germinated seedlings of Cymodocea nodosa were used as a source of young embryonic tissue for cell culture. Histological study of the cotyledon revealed the presence of meristematic tissue that gave rise to the first leaf of the plantlet, which released large numbers of cells after enzymatic digestion and thus indicated great growth potential. An effective enzy- matic digestion protocol for C. nodosa meristem tissue was developed; this consisted of a 24-h digestion period at 28 ° C using a 0.6- " enzymatic solution (plasmolysis medium containing 1 % (w:v) cellulase and hemicel- lulase). It was necessary to add citric acid (0.02 " as an antioxidant to the enzymatic solution to obtain the highest cell yield and viability (mean ± SE: 371.6 ± 30.1 ! 10 5 cells g -1 fw and 97.8 ± 0.6 % , respectively). NaCl and other salt components of the seawater were key factors in the establishment of axenic cell cultures and their further progress; their absence in the medium inhibited contami- nant overgrowth. Nevertheless, cells maintained growth for 90 days, even without hormones and deprived of salts, although these salts might be necessary to allow the development of morphogenetic programmes desired for plant production. In this manner, we generated a basic tool for the future of C. nodosa in vitro culture that will allow the establishment and maintenance of hormone- autonomous cell cultures with strong potential in future biotechnology for marine higher plants.