Agarose-induced embryoid formation in sunflower protoplasts is triggered by RGD-mediated membrane-matrix adhesion

Embedding of plant cells or protoplasts in gelling medium is widely used to improve cell viability and plating efficiency (Smidsrod and Skjak-Braek, 1990). In Arabidopsis thaliana (O’Neill and Mathias, 1993) and Helianthus annuus (Chanabe et al. 1991), protoplast inclusion in a solid matrix induces changes in division pattern and development. In sunflower, protoplasts cultured in liquid medium divide symmetrically and form loose microcolonies. In contrast, when they are embedded in agarose, most of them divide asymmetrically and display a polar organization at the onset of their development giving rise to compact embryo-like structures (Petitprez et al, 1995). These embryoids-progress to the heart-shape stage but do not develop further. Various hypotheses have been proposed to explain the effect of embedding on the fate of protoplasts: (i) limiting oxygen diffusion (Barbotin et al., 1993), (ii) simulating a cell wall, (iii) generating physical constraints that affect the stability of the cells (Asano et al., 1994). We previously showed that an increase of physical constraints such as hydrostatic pressure 0.4–1 MPa had an inhibitory effect on microtubule organization and cell wall synthesis and consequently on cell division (Barthou et al., 1997) but had no effect on division asymetry. Thus, physical constraints cannot solely explain the protoplast division pattern observed in agarose cultures.