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Expression of the vhb gene, encoding the hemoglobin protein from Vitreoscilla spp. (VHb), has been shown to increase cell growth and protein synthesis, modify the oxygen-dependent product biosynthesis and the susceptibility to oxidative and nitrosative stresses in several host microrganisms, and to improve plant tolerance to flooding-submergence. A chimeric construct consisting of the CaMV35S promoter fused to the vhb gene and nopaline synthase terminator was transferred into white poplar ( Populus alba L.) via Agrobacterium tumefaciens in order to test the generality of these phenomena. The presence of the vhb gene was demonstrated by Southern blot analysis. Accumulation of the vhb transcript and protein was detected in all the selected transgenic poplar lines. In vitro growth bioassays revealed that the vhb gene expression in transgenic poplar plants did not significantly affect their growth pattern. One out of the six selected transgenic lines showed significantly higher values for plant height and stem biomass in greenhouse conditions and exhibited enhancement of root biomass production and stem diameter when compared to the wild-type plants. However, no significant differences in chlorophyll a, b, total carotenoid and protein contents were observed. Two selected transgenic lines were characterized in more detail for tolerance to submergence, oxidative and nitrosative stresses. Under in vitro and in vivo submergence conditions, growth parameters and total protein content of transgenic VHb poplars were similar to those observed in the wild-type plants. In addition, leaf discs from the transgenic plants maintained in standard growth conditions did not reveal increased tolerance to oxidative stress by hydrogen peroxide compared to wild-type plants. In a parallel study, cell suspension cultures obtained from both wild-type and VHb transgenic lines were evaluated for growth and survival in the presence of oxidative and nitrosative stresses. No significant differences were observed between the tested VHb and wild-type poplar lines. Our results show that VHb expression in plants can have erratic effects since the enhancement of plant growth and biomass production and the tolerance to submergence, oxidative and nitrosative stresses are not consistently dependent on the presence of this specific function. Consequently, the genetic manipulation of plant oxygen metabolism must be carefully evaluated and extensive biochemical, molecular and cellular investigations are required to assess the real value of the final products. [ABSTRACT FROM AUTHOR]

RAPD (Random Amplified Polymorphic DNA) fingerprints have recently been used to estimate genetic and taxonomic relationships in plants. In this study RAPD analysis was performed on 32 clones belonging to different species of the genus Populus. Of these, 25 clones are registered in several countries for commercial use and, altogether, cover almost 50% of the worlds cultivated poplars. DNA was prepared from leaves and amplified by PCR using random oligonucleotide primers. Amplification products were separated by agarose-gel electrophoresis to reveal band polymorphisms. Four primers out of the 18 tested, were selected on the basis of the number and frequency of the polymorphisms produced. With these a total of 120 different DNA bands were reproducibly obtained, 92% of which were polymorphic. The polymorphisms were scored and used in band-sharing analyses to identify genetic relationships. With a few but interesting exceptions, these are consistent with the present taxonomy of the genus Populus and with the known predigrees of cultivated poplars. Moreover, the results show that RAPD analysis allows one to discriminate among all tested clones and can, therefore, be recommended as a convenient tool to defend plant breeders rights. [ABSTRACT FROM AUTHOR]

Two clones of Populus nigra L. were tested in vivo and in vitro for their susceptibility to three different Agrobacterium tumefaciens wild-type strains evaluating number and size of resulting calluses. Strain C58 proved to be the most virulent. Various parameters affecting Agrobacterium-mediated transformation of P. nigra clones were further analyzed using ß-glucuronidase gene transient expression. The clone Jean Pourtet proved to be more susceptible than the clone San Giorgio. A. tumefaciens strain A281 pKIWI105 proved to be the most virulent. The optimal procedure involved dipping of leaf discs into a bacterial suspension (7×10 cells/ml) for 20 min, followed by a 48 h co-cultivation period on semi-solid regeneration medium. Leaf explants were co-cultivated with two disarmed A. tumefaciens strains. Plantlets of San Giorgio were regenerated, tested for ß-glucuronidase activity and rooted on selective medium containing kanamycin. Polymerase chain reaction analysis and Southern blot hybridization confirmed the integration of the neomycin phosphotransferase II gene into the poplar genome. [ABSTRACT FROM AUTHOR]