Studies on cell wall regeneration in protoplast culture of legumes - the effect of organic medium additives on cell wall components

The cell wall regeneration in mesophyll protoplasts of yellow lupin and grass pea was studied. The occurrence of cell wall components: cellulose, callose and arabinogalactan proteins was analysed during 15 days of culture. Protoplasts were cultured in different media to test the effect of culture environment on the cell wall regeneration. Medium supplementation with 2 mg/l chitosan resulted in prolonged viability, more balanced cellulose resynthesis, increased callose formation and induction of mitotic divisions in protoplast-derived cells of both examined legumes. In chitosan-enriched medium arabinogalactan proteins were detected in cell plates of divided cells. Medium rich in additional organic compounds, i.e. free amino acids, organic acids and monosaccharides, was inferior to media of simpler composition. In both species the relatively quick cellulose resynthesis negatively affected the viability of protoplast-derived cells. In grass pea cellulose appeared during 24 h of culture. In yellow lupin the process started significantly later and after 10 days the frequency of walled cells did not exceed 50%. Callose was detected in cultures of both species and its pattern suggested that the synthesis was unlikely to be a result of protoplast wounding. Arabinogalactan proteins were localized in cell walls of different types of cells: dividing, elongating, but predominantly in degenerating ones. Occurrence and organization of the cell wall components studied were discussed in relation to recalcitrance of grass pea and yellow lupin protoplasts.