| Abstract: | Pollen preparations usually show a high nuclease activity. Therefore, to avoid DNA degradation, co-cultures of pollen and Agrobacterium tumefaciens were evaluated as a new tool in gene transfer experiments. As a model system, Petunia pollen was co-cultured with an A. tumefaciens wild strain. The co-cultured pollen was used for pollination of Petunia flowers. The seeds obtained were germinated and one cotyledon per seedling was removed and put stalk upwards on nutrient agar. In 80% of the cotyledons a callus developed from the cut surface of the stalks which was screened for tumor transformation on hormone-free medium. In repeated subculturing some calli maintained growth on hormone-free medium. Two of these calli were habituated. One callus, the best growing one, showed on Southern blot analysis a distinct hybridization signal at 3.2 kb when probed with Hind III fragment 22 DNA, covering two genes responsible for hormone free growth. This is the exact size that could be expected when plant material has been transformed with T-DNA. Another callus gave a hybridization signal at 2 kb which could only be explained with chromosomal rearrangement. In these two calli there was no co-transformation of the nos-gene: nopaline synthase activity could be detected from none of the calli, and none of the calli DNAs hybridized when probed with the nos-gene. Bacterial contamination could be excluded by probing the DNAs with virfragments. |
