Lipopolysaccharide and flagellin of Azospirillum brasilense Sp7 influence callus morphogenesis and plant regeneration in wheat.

In vitro somatic callus culturing is used widely in plant biotechnology, but its effectiveness depends largely on the donor plant genotype. Bacteria or components of their cells are rarely used to activate morphogenesis. In this work, inoculation of explants from immature wheat (Triticum aestivum L.) embryos with a suspension of living cells of the bacterium Azospirillum brasilense Sp7 resulted in callus death after 7 days of growth, in contrast to explant treatment with a suspension of heat-killed whole cells of Sp7. The experiments used two wheat lines, LRht-B1a and LRht-B1c, which differ in morphogenic activity. Growing calluses with the lipopolysaccharide of A. brasilense Sp7 increased the yield of regenerated plants 2- to 3.5-fold in both lines. This increase was through the activation of regenerant formation from morphogenic calluses. We have demonstrated for the first time the effects of bacterial flagellin on plant tissue culture. The polar-flagellum flagellin of A. brasilense Sp7 leveled the genotypic differences in the morphogenic ability of callus tissue. Specifically, it increased the yield of morphogenic calluses in the weakly morphogenic line LRht-B1a to the yield value in the highly morphogenic line LRht-B1c but lowered the yield of regenerants in the highly morphogenic line LRht-B1c to the yield value in the weakly morphogenic line LRht-B1a. Thus, bacterial lipopolysaccharides and flagellins can be used to regulate the formation of morphogenic calluses and regenerants in plant tissue culturing in vitro.

Effect of bacterial lipopolysaccharides on morphogenetic activity in wheat somatic calluses.

We evaluated the effect of lipopolysaccharides from the plant-growth-promoting associative bacterium Azospirillum brasilense Sp245 and from the enteric bacterium Escherichia coli K12 on the morphogenic potential of in vitro-growing somatic calluses of soft spring wheat (Triticum aestivum L. cv. Saratovskaya 29). A genetic model was used that included two near-isogenic lines of T. aestivum L. cv. Saratovskaya 29 with different embryogenic capacities; one of these lines carries the Rht-B1 dwarfing gene, whereas the other lacks it. When added to the nutrient medium, the lipopolysaccharide of A. brasilense Sp245 promoted the formation of calluses with meristematic centers and stimulated the regeneration ability of the cultured tissues in both lines. By contrast, the lipopolysaccharide of the enteric bacterium E. coli K12 barely affected the morphogenetic activity of callus cells and the yield of morphogenic calluses and regenerated plants. These findings indicate that the lipopolysaccharide of the plant-growth-promoting associative bacterium A. brasilense Sp245 specifically enhances the morphogenetic activity of wheat somatic tissues, which increases the efficacy of culturing of genotypes with a relatively low morphogenic potential. The results of the study may contribute to the improvement of the efficacy of plant cell selection and gene engineering and to a better understanding of the mechanisms responsible for plant recognition of lipopolysaccharides of associative bacteria.