Ultrastructure and histochemical analysis of extracellular matrix surface network in kiwifruit endosperm-derived callus culture

The study used Actinidia deliciosa endosperm-derived callus to investigate aspects of the morphology, histology and chemistry of extracellular matrix (ECM) structures in morphogenically stable tissue from long-term culture. SEM showed ECM as a membranous layer or reticulated fibrillar and granular structure linking the peripheral cells of callus domains. TEM confirmed that ECM is a distinct heterogeneous layer, up to 4 mum thick and consisting of amorphous dark-staining material, osmiophilic granules and reticulated fibres present outside the outer callus cell wall. ECM covered the surface of cells forming morphogenic domains and was reduced during organ growth. This structure may be linked to acquisition of morphogenic competence and thus may serve as a structural marker of it in endosperm-derived callus. ECM was also observed on senescent cells in contact with the morphogenic area. Treatment of living calluses with chloroform and washing with ether-methanol led to partial destruction of the extracellular layer. Digestion with pectinase removed the membranous layer almost completely and exposed thick fibrillar strands and granular remnants. Digestion with protease did not visibly affect the surface layer. Indirect immunofluorescence showed low-methylesterified pectic epitopes labelled by JIM5 monoclonal antibody. Immunolabelling, histochemistry, and solvent and enzyme treatments suggested pectins and lipids as components of the surface layer. These compounds may indicate protective, water retention and/or cell communication functions for this external layer.