Mirna Ćurković Perica

Homology-dependent DNA transfer from plants to a soil bacterium under laboratory conditions: implications in evolution and horizontal gene transfer.

DNA transfer was demonstrated from six species of donor plants to the soil bacterium, Acinetobacter spp. BD413, using neomycin phosphotransferase (nptII) as a marker for homologous recombination. These laboratory results are compatible with, but do not prove, DNA transfer in nature. In tobacco carrying a plastid insertion of nptII, transfer was detected with 0.1 g of disrupted leaves and in oilseed rape carrying a nuclear insertion with a similar quantity of roots. Transfer from disrupted leaves occurred in sterile soil and water, without the addition of nutrients. It was detected using intact tobacco leaves and intact tobacco and Arabidopsis plants in vitro. Transfer was dose-dependent and sensitive to DNase, and mutations in the plant nptII were recovered in receptor bacteria. DNA transfer using intact roots and plants in vitro was easily demonstrated, but with greater variability. Transfer varied with plant genome size and the number of repeats of the marker DNA in the donor plant. Transfer was not detected in the absence of a homologous nptII in the receptor bacteria. We discuss these results with reference to non-coding DNA in plant genomes (e.g., introns, transposons and junk DNA) and the possibility that DNA transfer could occur in nature.

Separation of hypoviral double-stranded RNA on monolithic chromatographic supports

A procedure based on BIA Separations CIM DEAE anion-exchange chromatography was developed to separate double-stranded (ds) RNA of hypovirus infecting phytopathogenic fungus Cryphonectria parasitica. Using a linear gradient of 25 mM 4-morpholinepropanesulfonic acid (MOPS), pH 7.0 as a binding buffer, and 25 mM MOPS, 1.5 M NaCl, 0.1 mM EDTA, 15% isopropanol (v/v), pH 7.0 as an elution buffer, hypoviral dsRNA was additionally purified from nucleic acid species present in preparations partially purified by standard CF-11 cellulose chromatography. Moreover, crude phenol/chloroform extracts of the fungal tissue were also applied to monolithic supports and CIM DEAE chromatograms revealed clear evidence for hypoviral presence without CF-11 chromatography, nucleic acid precipitation, and electrophoresis.

In vitro propagation of Inula verbascifolia (Willd.) Hausskn. subsp. verbascifolia

Abstract A clonal propagation method has been developed for Inula verbascifolia (Willd.) Hausskn. subsp. verbascifolia, a species of horticultural as well as potential medicinal interest. Shoots originating from aseptically germinated seeds were used for culture initiation. The highest multiplication rate of 6.5 shoots per explant was achieved in an 8-week culture period on Murashige and Skoog medium supplemented with 2.2 μM 6-benzylaminopurine and 2.9 μM gibberellic acid. During the first three subcultures of the multiplication phase, two-thirds of explants spontaneously rooted on all tested media. Rooting of excised shoots on MS medium supplemented with 3 μM IAA or 3 μM IBA was 100%. Aside from the fact that all the explants were rooted on this root-inducing media, explants developed a surprisingly high mean number of shoots, 4.6 and 2.9 after 4 weeks, on medium supplemented with IAA or IBA, respectively. Rooted plantlets were transferred to potting soil and acclimatized to outdoor conditions.