“PLANT CELL PROLIFERATION INSIDE AN INORGANIC MATRIX”

MERCEDES PERULLINI; MATÍAS JOBBÁGY; MARÍA MERCEDES RIVERO; ALEJANDRO MENTABERRY

Pucón, Chile

Simposio; 9th International Symposium on Biomineralization; 2005

CIMAT

The sol-gel encapsulation of whole cells within inorganic matrices is a subject of great scientific and technological interest. Biologically modified ceramics (biocers) for a wide range of applications have been described.1 In conventional encapsulation techniques cells are permanently immobilized and are not able to proliferate within the matrix.2 This limitation excludes the possibility of applying such devices to the production of secondary metabolites in bioreactors. In this work, we use a novel strategy3 for plant cell immobilization allowing cell division and growth inside inorganic matrices. Plant cell cultures used in these experiments were initiated from BY2 tobacco calli and from carrot phloematic tissue.4 Cell suspensions were obtained from calli grown under controlled conditions on semi-solid Murashige and Skoog (MS).5 Stock cell suspension cultures were grown under orbital agitation (120 rpm) at (24±1) °C.  The protocol used for biocers synthesis involved previous immobilization of plant cells in an organic Ca-alginate gel to avoid any harmful contact with silica precursors [sodium silicate 0.63 M, colloidal silica (Ludox 40 HS, Sigma-Aldrich) and succinic acid (1.5 wt)]. Once immobilization was performed, a semi-rigid monolith was obtained. The cells were included inside the monolith macrocavities containing liquid MS culture media. Then, the monoliths were kept under the same culture conditions as stock cell suspensions. Following the encapsulation procedure, initial cell survival and subsequent cell growth, were monitored by staining with Fluorescein diacetate-propidium iodide (FDA-PI). This procedure proved to be biocompatible for the two cell types assayed, since almost 100% of the encapsulated cells remained metabolically active. Cellular proliferation inside the inorganic matrix was monitored along a 6-month period. Fluorescence microscopy images taken at different times post-immobilization revealed the presence of active cell division and formation of cell clusters inside the cavities. After 6 months, plant calli of up to 1 mm in diameter could be observed inside each macrocavity. To assess the effectiveness of the silica matrix as a barrier against contamination, culture medium was inoculated with E. coli. Bacterial growth was analyzed after 48 h by plating samples taken from the cavities and the external culture media on Triptein-Soya semi-solid medium. E. coli was detected exclusively in the external medium samples. Monoliths obtained showed good mechanical stability, suggesting that they could be used in future applications for long-term plant cell entrapment. Notes and references 1. Böttcher, H.; Soltmann, U.; Mertig, M.; Pompe, W. J. Mater. Chem., 14:2176, 2004. 2. Pressi, G.; Dal Toso, R.; Dal Monte, R. J. Sol-Gel Sci. Tech. 26:1189, 2003. 3. Perullini, M.; Jobbágy, M.; Soller-Illia, G. J. A. A.; Bilmes, S. A. Chem. Mater. 17:3806, 2005. 4. Nagata, T; Nemoto, Y; and Hasezawa, S. Int. Rev. Cytol. 132:1-30, 1992; Tatsuhito Fujimura, T. and Komamine, A. Plant Physiol. 64:162–164, 1979. 5. Murashige, T. and Skoog, F. Physiol. Plant. 15:473-497, 1962.