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.

Structure of the polysaccharides from the lipopolysaccharide of Azospirillum brasilense Jm125A2.

Two polysaccharides were obtained by mild acid degradation of the lipopolysaccharide of associative nitrogen-fixing bacteria Azospirillum brasilense Jm125A2 isolated from the rhizosphere of a pearl millet. The following structures of the polysaccharides were established by sugar and methylation analyses, Smith degradation, and (1)H and (13)C NMR spectroscopy: [Formula: see text] Structure 1 has been reported earlier for a polysaccharide from A. brasilense S17 (Fedonenko YP, Konnova ON, Zdorovenko EL, Konnova SA, Zatonsky GV, Shaskov AS, Ignatov VV, Knirel YA. Carbohydr Res 2008;343:810-6), whereas to our knowledge structure 2 has not been hitherto found in bacterial polysaccharides.

Structural studies of the polysaccharides from the lipopolysaccharides of Azospirillum brasilense Sp246 and SpBr14.

Lipopolysaccharides from closely related Azospirillum brasilense strains, Sp246 and SpBr14, were obtained by phenol-water extraction. Mild acid hydrolysis of the lipopolysaccharides followed by GPC on Sephadex G-50 resulted in polysaccharide mixtures. On the basis of sugar and methylation analyses, Smith degradation and (1)H and (13)C NMR spectroscopy data, it was concluded that both bacteria possess the same two distinct polysaccharides having structures 1 and 2: [structure: see text]. Structure 1 has been reported earlier for a polysaccharide of A. brasilense 54 [Fedonenko et al., 2011] whereas to our knowledge structure 2 has not been hitherto found in bacterial polysaccharides.

Structural studies of the O-specific polysaccharide(s) from the lipopolysaccharide of Azospirillum brasilense type strain Sp7.

Lipopolysaccharide was obtained by phenol-water extraction from dried bacterial cells of Azospirillum brasilense type strain Sp7. Mild acid hydrolysis of the lipopolysaccharide followed by GPC on Sephadex G-50 resulted in a polysaccharide mixture, which was studied by composition and methylation analyses, Smith degradation and (1)H and (13)C NMR spectroscopy. The following polysaccharide structures were established, where italics indicate a non-stoichiometric (∼40%) 2-O-methylation of l-rhamnose.

Structure of repeating units of a polysaccharide(s) from the lipopolysaccharide of Azospirillum brasilense SR80.

A high-molecular mass polysaccharide fraction was obtained by mild acid hydrolysis of the lipopolysaccharide of diazotrophic rhizobacterium Azospirillum brasilense SR80 followed by GPC on Sephadex G-50 Superfine. Studies by composition and methylation analyses, Smith degradation, and 1D and 2D (1)H and (13)C NMR spectroscopy demonstrated the presence of two structurally distinct repeating units having the following structures: It seems likely, although not proved, that these are repeats of two distinct polysaccharides rather than they build blocks within the same polysaccharide chain. The former structure is new, whereas the latter is closely related to the O-polysaccharide structure of A. brasilense Jm6B2 established earlier, which differs in partial (~60%) 3-O-methylation of d-rhamnose only [Boiko, A. S.; Dmitrenok, A. S.; Fedonenko, Yu. P.; Zdorovenko, E. L.; Konnova, S. A.; Knirel, Y. A.; Ignatov, V. V. Carbohydr. Res.2012, 355, 92-95].