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 - 5.01

GENETIC TRANSFORMATION OF STRAWBERRY BY USING A REPEATED VIRAL GENE AS A MODEL FOR VIRUS RESISTANCE

R. DONNOLI, S. COMES, A. FANIGLIUOLO, A. CRESCENZI, I. gRECO, G. MARTELLI, F. SUNSERI

Dipartimento di Biologia, Difesa e Biotecnologie AgroForestali, Università degli Studi della Basilicata Contrada Macchia Romana, 85100 Potenza

Fragaria x ananassa, Agrobacterium tumefaciens

The genetic transformation of the cultivated strawberry (Fragaria x ananassa Duch.) have been yet reported (Orlando et al. 1997, Plant Cell Rep. 16: 272-276; Barcelo et al. 1998, Plant Cell Tiss. Organ Cult. 54: 29-36; de Mesa et al. 2000, Aust. J. Plant Physiol. 27: 1093-1100; Ricardo et al. 2003, HortSci. 38: 277-280). Since different transformation strategies have been developed, the techniques are highly genotype-dependent and the transformation frequencies obtained are frequently below 5%. As recently reported by de Mesa et al. (2000) combined techniques (wounding tissue before Agrobacterium infection, by means of particle gun) could improve the transformation efficiency.

On the other hand there are different viruses infecting Fragaria species. Strawberry mottle virus (SMoV) is the most widespread virus worldwide. In single infections, yield losses of up to a 30% have been reported. In mixed infections with, for example, Strawberry crinkle virus SCV), Strawberry vein banding virus (SVBV) and/or Strawberry mild yellow edge virus (SMYEV), the losses can be even higher. Historically, SMoV was often considered to be a mild strain of SCV, and it was not until the mid-1950s that SMoV symptoms were generally accepted as being caused by an aetiologically distinct virus. All the Fragaria species are susceptible to SMoV, though many are symptomless hosts that eventually have reduced vigour and yield depending on the severity of the strain (Mellor & Krczal, 1987, In: Virus Diseases of Small Fruits, USDA Agricultural Handbook 631, pp. 10-16).

Three strawberry cultivars (Clea, Irvine, Paros) useful for South Italy conditions and four accessions of Fragaria vesca, coming from Southern Italy areas, were utilized for genetic transformation by using Agrobacterium-mediated techniques. Four different MS-based media were tested in order to obtain callus proliferation and shoot regeneration. Leaf disks were also co-cultivated with a LBA 4404 Agrobacterium tumefaciens containing a plasmid with the selectable marker gene NPTII, a vital marker like the Green Fluorescent Protein (GFP, Chalfie et al. 1994, Science 263: 802-805) and a repeated viral gene. The infection was performed in a vacuum chamber for 20 minutes.

High percentage (12%) of organogenesis and plant regeneration after Agrobacterium cocultivation were obtained in strawberry (cv “Paros”) by using a MS medium supplemented with 5 µM IBA and 5 µM BAP and the plasmid harbouring the CP of TSWV. A sufficient number of genetic transformation events were obtained either in strawberry (6%) or in Fragaria vesca (2%) by using the plasmid harbouring the GFP marker. Rarely, genetic transformation events were obtained also by particle bombardment, than the combined transformation technique (de Mesa et al. 2000) would to be tested.

Nevertheless, the genetic transformation protocol for the strawberry genotypes utilized is now optimized, while several experiments will be carry out for the diploid species.

In the present study, a highly efficient and reliable protocol for plant regeneration and interesting results of Agrobacterium-mediated transformation in strawberry have been reported. The utilization of a repeated viral gene putative responsible for gene silencing was considered for modelling the analysis of virus infection in strawberry, since the Fragaria species was reported as sympotmless hosts of several viruses.