Marzena Popielarska-Konieczna

Extracellular matrix of plant callus tissue visualized by ESEM and SEM.

Actinidia deliciosa endosperm-derived callus culture is stable over a long period of culture. This system was used to investigate the ultrastructure of extracellular matrix occurring in morphogenic tissue. Specimens were prepared by different biological techniques (chemical fixation, liquid nitrogen fixation, glycerol substitution, critical-point drying, lyophilization) and observed by scanning electron microscopy (SEM). Fresh and wet samples were analyzed with the use of environmental scanning electron microscopy (ESEM). Extracellular matrix was observed on the surface of cell clusters as a membranous layer or reticulated network, shrunken or wrinkled, depending on the procedure. Generally, shrunken membranous layers with a globular appearance and fibrils were noted after critical-point drying and liquid nitrogen fixation. Smoother surface layers without visible fibrils and showing porosity were typically seen by environmental scanning electron microscopy. Preservation with glycerol substitution caused wrinkled appearance of examined layer. Analysis of fresh samples yielded images closer to their natural state than did critical-point drying or fixation in liquid nitrogen, but it seems best to compare the results of different visualization methods. This is the first report of ESEM observations of plant extracellular matrix and comparison with SEM images from fixed material.

Are extracellular matrix surface network components involved in signalling and protective function

Endosperm is an interesting model for in vitro experiments, because of its unique origin, development and ploidy level. Here we used Actinidia deliciosa endosperm-derived callus to investigate morphology, histology and chemistry of extracellular matrix (ECM) structures in morphogenically stable tissue from long - term culture. SEM and TEM analysis showed that ECM is a heterogenous layer which consists of amorphous, dark – staining material, osmiophilic granules and reticulated fibres outside the outer callus cell wall. This structure may serve as a structural marker of morphogenic competence in endosperm – derived callus, because of its presence on the surface of callus forming morphogenic domains and its disappearance during organ growth. Based on immunolabelling, histochemistry, solvent and enzyme treatments, we suggest that pectins and lipids are components of the ECM layer. These results might indicate protective, water retention and/or cell communication functions for this ECM layer. Addendum to: Popielarska-Konieczna M, Kozieradzka-Kiszkurno M, Świerczyńska J, Góralski G, Ślesak H, Bohdanowicz J. Ultrastructure and histochemical analysis of extracellular matrix surface network in kiwifruit endosperm-derived callus culture. Plant Cell Rep 2008; 27:1137-45.

The effect of genotype on a barley scutella culture. Histological aspects

Cereals are known to be recalcitrant to the induction of morphogenesis in vitro and the majority of the methods used are callus-mediated and species/genotype-dependent. In the present investigation, a method of morphogenesis induction from immature scutella of selected barley cultivars was used, and particular attention was paid to histology in the initial stages of the regeneration process in order to confirm whether it occurs directly or indirectly (via callus formation). The length of the period from inoculating scutella on the medium to obtaining plantlets depended on the cultivar and the individual scutellum of the barley and varied between 2.5–4 months. The regeneration efficiency and viability of barley scutella was revealed to be highly genotype dependent. The average number of regenerated plants per regenerating scutellum was highest in the case of cv Granal (3.7). A histological analysis of the cultured explants showed both non-morphogenic and morphogenic callus formation. Two types of indirect morphogenetic response were observed: organogenesis (shoot bud formation) and somatic embryogenesis. This is the first report concerning an analysis of in vitro regeneration from immature scutella of barley cultivars (Stratus, Ryton, Granal and Binal).