Erysiphe necator overwinters as ascospores in cleistothecia and mycelium in dormant buds of grapevines. Shoots developing from infected buds early in the growing season are covered with dense mycelium and are known as "flag shoots". Combining epidemiological and genetic analyses, the objective of this study was to analyze the spatial and genetic structure of a flag shoot subpopulation of E. necator as a way to assess the contribution of flag shoots as primary inoculum, and to determine if flag shoot subpopulations are clonal with only one mating type. One vineyard in Tuscany, Italy was surveyed intensively for flag shoots for 8 years; isolations of E. necator were made from flag shoots for 5 years. We observed distinct disease foci developing around flag shoots early in epidemics, demonstrating a steep dispersal gradient of conidia and the importance of flag shoots as primary inoculum sources. Flag shoots were spatially aggregated within and between years, most likely as a result of short-distance dispersal of conidia from flags early in the season when dormant buds for the next year's shoots are formed and are susceptible to infection. The two mating types were found in 1:1 ratios in this flag shoot subpopulation. Genotypic diversity, based on inter-simple sequence repeat markers, was high in all years with only two haplotypes occurring twice, and subpopulations were genetically differentiated between years. Similarities between haplotypes were not spatially autocorrelated. One multilocus analysis of population structure is consistent with the hypothesis of random mating but another is not. These results are not consistent with expectations for a strictly clonal or strictly randomly mating flag shoot subpopulation. Instead, the hypothesis that the flag shoot subpopulation of E. necator may reproduce clonally and sexually needs further testing.

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The presence of T-DNA was examined by Southern blot analysis in 16 regenerated shoot lines derived from 6 Agrobacterium rhizogenes-transformed root clones of Solanum tuberosum L. cv. Bintje. TR-DNA, present in regenerated shoot lines from 3 out of 6 root clones was correlated with the presence of opines. One root clone produced opines up to 2.5 years of subculture. However, plant regeneration from and prolonged subculturing of this root clone resulted in loss of opine synthesis, caused by deletion of TR-DNA. TL-DNA inserted at 1 to 5 independent loci was found in 14 of the 16 shoot lines. Surprisingly, 1 to 2 additional insertions next to similar insertions of TL-DNA were found in shoot lines from the same root clone (named 'sister' shoot lines) in 2 out of 4 root clones. Nevertheless, this did not result in gross phenotypic variation between sister shoot lines. Another root clone regenerated 1 shoot line with an Ri phenotype, containing 1 insertion of TL-DNA, and 2 shoot lines with a normal Bintje phenotype without TL-DNA. The 5th root clone showed no difference between sister shoot lines and the 6th root clone produced only 1 shoot line. We conclude that during prolonged root culture and during shoot regeneration from root clones deletion of TL- and TR-DNA insertions can occur. The significance of the frequency of deletion of T-DNA of the Ri plasmid is discussed.

Agrobacterium rhizogenes transformed and control roots of the tetraploid potato cv. Bintje were compared. Transformed roots were obtained after infection by A. rhizogenes 15834 or 1855. Both in leaf and stem segments, more roots were formed at the basal side of the segments, indicative for a polarity in root formation. As compared to control roots the transformed roots are characterized by smaller and more densely stained cells, a zone of cell division, and smaller statoliths. These characteristics are correlated with vigorous growth, high branching incidence and diminished geotropism. The plant regeneration procedure according to Ooms et al.[1] was modified. The transformed roots required less 2,4-D than control roots for the induction of shoot-competent calli. The callus and shoot induction phases were reduced from 8 and 6 weeks to 3 and 3 weeks, respectively. Upon induction, 25%, 58% and 61% of the root clones originating from tuber, stem and leaf, respectively, produced shoots, whereas all of the control roots produced shoots. Shoot outgrowth occurred on liquid MS medium in the absence of hormones.

The Dutch potato cultivar Bintje has been transformed by Agrobacterium strain LBA1060KG, which contains two plasmids carrying three different DNAs (TL- and TR-DNA on the Agrobacterium rhizogenes plasmid and TKG-DNA on the pBI121 plasmid). Several transformed root clones were obtained after transformation of leaf, stem, and tuber segments, and plants were then regenerated from these root clones. The expression of the various marker genes [rol, opine, β-glucuronidase (GUS), and neomycin phosphotransferase (NPTII)] was determined in several root clones and in regenerated plants. The selection of vigorously growing root clones was as efficient as selection for kanamycin resistance. In spite of the location of NPTII and GUS genes on the same T-DNA, 17% of the root clones did not show GUS activity. Nevertheless, Southern blot analysis showed that these root clones contained at least three copies of the GUS gene. Sixty-four per cent of the root clones contained opines. The expression of these genes, however, was negatively correlated with plant regeneration capacity and normal plant development. The differential expression of the marker genes in the transgenic potato tissues is discussed.