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 - 1.21

THE USE OF PHOSPHOMANNOSE ISOMERASE AS A SELECTABLE MARKER TO TRANSFER FOREIGN GENES IN GRAPE (VITIS SPP.)

SEMENZATO M., POLETTI V., MARTINELLI L.

Istituto Agrario San Michele all’Adige, 38010 San Michele all’Adige (TN)

selection marker gene, phosphomannose isomerase, Vitis

As for other plants, a valuable system for grape genetic transformation requires not only efficient gene delivery and efficient regeneration, but also an efficient selection system. In grape, the mostly applied strategy for selecting tissues after co-culture with Agrobacterium tumefaciens is the resistance induction to the antibiotic kanamycin obtained with the NPTII gene, encoding for neomycin phosphotransferase. In this system, only transformed cells are able to survive in the culture when proper kanamycin concentration is provided in the medium. The recent concern on the use of antibiotic resistance in transgenic plant production, and the aim to employ constructs at low ecological impact, is addressing researchers to develop gene delivering systems based on various marker genes. Within these systems, mannose is being used as a selectable agent and the phosphomannose isomerase (PMI) gene from E. coli as marker. Not all plants can naturally utilize mannose as carbon source, while PMI-transformed cells become able to grow with the addiction of no or only small amounts of sucrose. Thus, as a consequence of a positive selection, on mannose-containing medium only PMI-expressing cells proliferate within the subcultures. The system, licensed by Syngenta Company, resulted valuable in various herbaceous plant species, while it was never attempted in grape.

With the aim of assay marker genes at low ecological impact during grape genetic transformation, we are evaluating mannose as selection agent in several Vitis genotypes, all of them with peculiar in vitro performances. Before transferring the PMI gene, a patient and huge preliminary work was required for choosing the most suitable sucrose/mannose combinations. Embryogenic cultures at different morphogenic development of the V. vinifera cvs. Chardonnay and Brachetto a grappolo lungo, the genotype V. rupestris and the rootstock Kober 55B were subcultured for several months on media added with different sucrose and mannose concentrations. Visible different grow capabilities were exhibited by the several cultures in the various media tested.

First genetic transformation assays were performed; embryogenic cultures were co-cultured with Agrobacterium tumefaciens strain LBA4404 carrying the Syngenta construct pNOV2819 which contains the E. coli PMI gene under a short-version of the promotor of the Cestrium Yellow Leaf Curling Virus. The effectiveness of this system in our grape cultures id under investigation.

This research was supported by the project OSSERVA of he Autonomous Province of Trento.