Morpho-anatomical characterization of mature embryo-derived callus of rice (Oryza sativa L.) suitable for transformation.

The objective of this study was to morpho-anatomically characterize embryogenic rice calli during early induction of somatic embryogenesis of three Brazilian rice cultivars. Herein, we explored embryogenic units (EUs) from 2-week-old cut proliferated calli to verify whether they were suitable for Agrobacterium tumefasciens-mediated transformation. Histological analysis and scanning electron microscopy (SEM) were used to analyze these types of calli during early rice callogenesis in the cultivars BRS Primavera, BRS Bonança, and BRS Caiapó. The characteristics of the embryogenic cells were preserved in the EUs, which showed a globular, compact structure that contained tightly packed cells and thus rendered the cells suitable for transformation. The EUs of BRS Caiapó also maintained the characteristics of the non-embryogenic callus, such as an elongated morphology and a lack of cellular organization. In general, the observations of the histological sections corresponded with those of the SEM images. The histological analysis suggested that all cultivars used in these experiments have morphogenic potential. The EUs from proliferated 2-week-old cut calli maintained their embryogenic features. The EUs were subjected to Agrobacterium-mediated transformation, which exhibited a regeneration frequency of 58 % for transformed hygromycin-resistant cell lines. These results show that EUs from proliferated 2-week-old cut calli are suitable for plant transformation.

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.