Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress

Giardini Naxos, Italy - 18/21 September, 2002

ISBN 88-900622-3-1

Poster Abstract - 3.19

MEASURING GENE FLOW FROM TWO BIRDSFOOT TREFOIL CULTIVATIONS BY USING TRANSGENES AS TRACER MARKERS

DE MARCHIS F.

Istituto di Ricerche sul Miglioramento Genetico delle Piante Foraggere, Via Pennetti Pennella, 22, 06128 Perugia, Italia

birdsfoot trefoil, pollen dispersal, gene flow, transgenic plants

Birdsfoot trefoil (Lotus corniculatus c.v. Leo, 2n = 24) is a perennial forage legume with high nutritive value used for pasture and hay. At this time, there are no data concerning the pollen diffusion from transgenic L. corniculatus field and the present study will clarify the possibility, in central Italy, of gene escape, the frequency at which this occurs and the eventual cross pollination between birdsfoot trefoil and two related species, L. tenuis and L. pedunculatus. Transgenic L. corniculatus plants were generated with Agrobacterium-mediated transformation. Two species of Agrobacterium were used: A. tumefaciens harboring the asparagine synthetase (asnA) gene and A. rhizogenes carrying the b-glucuronidase (uidA) gene. In the spring of 2001, in the experimental field located in Perugia, two pollinating plots, each one with 24 transgenic birdsfoot trefoil plants (24 transformed with the asnA gene and 24 with the uidA gene), were transplanted at the two opposite corners of a 64 x 40 m field. One beehive was located near each corner. Sixteen recipient plots were distributed in the whole area of the field and, in each plot, 4 wt plants for each of the three species considered were transplanted. During the same spring, in the experimental field in Rome, a 2 ha field trial was prepared to address the effect of field size on pollen. The pollinating plot consists of 200 transformed plants (100 with the asnA gene and 100 with the uidA gene). Ten recipients plots were distributed in the field. Two beehives were located near the source of transgenic pollen. Seeds were hand-harvested at the end of summer in both fields and were sown in the greenhouse for DNA investigation. Gene flow was evaluated by PCR. For the uidA gene, the X-Gluc staining assay was also performed to confirm data obtained from PCR amplification. Gene flow was measured as the percentage of plants with a transgene-specific PCR product with respect to the totality of plants analysed. It was observed that the horizontal transfer of trangenes from transgenic L. corniculatus plants to non transgenic plants can occurs only within the same species, while is completely absent between L. corniculatus and L. tenuis or L. pedunculatus. Interestingly, gene flow was observed in both field trials just for the asnA transgene, while it was no detectable for the uidA gene. These results seem to be associated to the sterility of uidA-expressing plants. Data obtained from the two field trials were markedly different likely due to the field size: while in Perugia the pollen diffusion was contained and occurred just for 18 meters away from the asnA pollinating plot with a frequency of 0,24%, in Rome the distance reached by the transgene is considerably higher, being the gene detectable till the distance of 120 m away from the source plot with a frequency of 0,74%. Interestingly, the gene flow was strictly associated with the position of the recipient plots with respect to the beehive in both fields. The transgene was inherited by progenies of plants placed in the recipient plots just in front of the pollinating one (as in Perugia), except if there was a barrier in front of the beehive. Honeybees, finding an obstacle straight on their way from and towards the colony domiciles, avoided it (as in Rome). The data collected demonstrate that birdsfoot trefoil pollen can travels and that the percentage of gene flow is far from to be zero also at considerable distances. To contain pollen spread, when bees are its vehicle, it would be useful to surround transgenic L. corniculatus fields with a cultivation of non transgenic plant belonging to a species with a superior height and sexually incompatible (as maize for example) which, consequently, would limit the pollination inside the boundaries of the birdsfoot trefoil field.