The Sinorhizobium fredii HH103 rkp-1 region, which is involved in capsular polysaccharide (KPS) biosynthesis, is constituted by the rkpU, rkpAGHIJ, and kpsF3 genes. Two mutants in this region affecting the rkpA (SVQ536) and rkpI (SVQ538) genes were constructed. Polyacrylamide gel electrophoresis and (1)H-NMR analyses did not detect KPS in these mutants. RT-PCR experiments indicated that, most probably, the rkpAGHI genes are cotranscribed. Glycine max cultivars (cvs.) Williams and Peking inoculated with mutants SVQ536 and SVQ538 showed reduced nodulation and symptoms of nitrogen starvation. Many pseudonodules were also formed on the American cv. Williams but not on the Asiatic cv. Peking, suggesting that in the determinate nodule-forming S. fredii-soybean symbiosis, bacterial KPS might be involved in determining cultivar-strain specificity. S. fredii HH103 mutants unable to produce KPS or exopolysaccharide (EPS) also showed reduced symbiotic capacity with Glycyrrhiza uralensis, an indeterminate nodule-forming legume. A HH103 exoA-rkpH double mutant unable to produce KPS and EPS was still able to form some nitrogen-fixing nodules on G. uralensis. Thus, here we describe for the first time a Sinorhizobium mutant strain, which produces neither KPS nor EPS is able to induce the formation of functional nodules in an indeterminate nodule-forming legume.

We developed procedures for in vitro cloning of Cedrus atlantica Manetti and C. libani A. Rich explants from juvenile and mature plants. Explant size was one determinant of the frequency of axillary bud break in both species. Shoot tips and nodal explants mainly developed calli, whereas bud sprouting occurred in defoliated microcuttings cultured on a modified Murashige and Skoog medium without growth regulators. Isolation and continuous subculture of sprouted buds on the same medium allowed cloning of microcuttings from C. atlantica and C. libani seedlings and bicentennial C. libani trees, thus providing a desirable alternative for multiplying mature trees that have demonstrated superior characteristics. We also report adventitious bud differentiation from isolated embryos of C. atlantica. Neither auxin treatments nor other methods tested, including infection with Agrobacterium rhizogenes, were effective in inducing root initiation.

Successful propagation of Nerium oleander L. (oleander) was achieved by in vitro methods. Shoot cultures were initiated from seedlings of wild-growing plants and from shoot apices of adult plants belonging to the commercial cultivars Splendens Giganteum, Revanche, and Alsace. Axillary shoot breaking from shoot tips excised from these cultures required the presence of either 6-benzylaminopurine (BA) or thidiazuron (TDZ). The higher number of axillary shoots from juvenile material was obtained by culturing shoot tips from BA-pretreated material derived from seedlings on a modified Schenk and Hildebrandt medium (SHM) supplemented with BA or TDZ (average of 3.9 shoots per explant with a mean length of 10.4 mm) and when the media were supplemented with 8.8 μM TDZ (average of 3.5 shoots per explant with a mean length of 7.3 mm) or 4.4 μM BA (average of 3.3 shoots per explant with a mean length of 12.3 mm). Among cultivars, cv. Revanche showed best shoot proliferation rates, especially when explants were cultured on Woody Plant Medium (average of 3.2 shoots per explant with a mean length of 10.2 mm). Adventitious bud differentiation from oleander leaves is also reported. Leaves excised from seedling-derived shoot cultures responded better than those from adult plant-derived shoot cultures (40% versus 5%, respectively). Bud differentiation required the presence of TDZ in the SHM medium, although shoot development was only achieved on transference of explants to media without TDZ but supplemented with BA and indoleacetic acid (IAA) or BA, kinetin, and IAA. Axillary and adventitious shoots were easily rooted (99%) and successfully (95% to 100%) transferred to soil.

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