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

Verona, Italy - 24/27 September, 2003

ISBN 88-900622-4-X

Poster Abstract - 5.30

STABLE PLASTID TRANSFORMATION OF POTATO CV. DESIREE WITH A GENE INVOLVED IN FATTY ACID DESATURATION

D. Gargano, W. Craig, P. Lenzi, N. Scotti, S. Grillo, T. Cardi

CNR-IGV, Institute of Plant Genetics, Res. Div. Portici

cardi@unina.it

plastid transformation, potato, transgenic plants, unsaturated fatty acids

In comparison with nuclear transformation, the transformation of the plastidial genome shows several advantages, such as: transgene containment caused by maternal inheritance of cytoplasmic organelles, precise transgene insertion in a defined plastome region by homologous recombination, as well as high and stable gene expression and protein accumulation. In crops other than tobacco, however, its efficiency and applicability are rather limited, and reports of successful transgene expression are still scanty. Herein we report the integration and expression of a Delta-9 desaturase gene, controlling the insertion of double bonds in fatty acid chains, in the plastidial genome of the widely used potato cv. Desiree. A higher content of unsaturated fatty acids is a desirable trait for stress tolerance of higher plants as well as for their nutritional value.

In the process of devising a plastid transformation protocol in potato we tested the regeneration potential from leaf explants of various genotypes using a number of either single or double-step regeneration protocols. The cv. Desiree showed profuse shoot proliferation on several media and thus was selected for transformation experiments. Optimal conditions for gene delivery using the biolistic approach were set up using different vectors and shooting parameters. In experiments with the nuclear vector pGUS-HYG (courtesy of Dr. T. Kavanagh, Ireland) up to 4.1 hpt⁺ shoots per bombardament were obtained.

As far as plastid transformation was concerned, the gene encoding a Delta-9 stearoyl-ACP desaturase, isolated from the wild potato species S. commersonii (Grillo et al. 1996 In: Physical Stresses in Plants, 163-169) was cloned into the plastid vector pZS197 (Svab and Maliga 1993 Proc. Natl. Acad. Sci. Usa 90, 913-917) and transferred in the potato plastome by the biolistic approach. Using a single step regeneration protocol and 500 mg l^-1 of the antibiotic spectinomycin for selection, we obtained seven explants with green calli from eight bombardaments; so far, two of them regenerated shoots. PCR analysis with primers annealing to the flanking regions of the inserted sequence revealed the right integration of the transgene and the complete homoplasmy of the transformed cells. Further, Northern analysis showed the correct transcription of the transgene. Western analysis is under way to verify the presence and the level of protein production in the plastid.