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

Salsomaggiore Terme, Italy - 26/29 September, 2001

ISBN 88-900622-1-5

Oral Communication Abstract

MARKER FREE TRANSGENIC PLANTS BY CO-CULTIVATION WITH TWO A. TUMEFACIENS STRAINS

BERIO T., FILIDEI E., GIOVANNINI A., MORREALE G., ALLAVENA A.

Istituto Sperimentale per la Floricoltura, C. so Inglesi 508, 18038 Sanremo, Imperia

attP, co-expression, co-segregation, co-transformation, cre/loxP

Transgenic tobacco plants were obtained following co-cultivation with two A. tumefaciens strains carrying the pGreen/pSoup vector system. Six independent, transformed T1 genotypes expressing both the selectable (NPTII) and the screenable (35SGUS) marker genes, were recovered on selective, medium. In two transformation experiments the co-expression frequency was 5.2 and 9.3%. respectively. Histochemical assay showed that the 35SGUS gene segregate following a 3:1 ratio in the progenies of five T1 genotypes and following a 15:1 ratio in a progeny. Data regarding regeneration on kanamicine selective medium demonstrate that the NPTII gene segregation was 3:1 and 15:1 in three and one T2 progenies respectively; two T2 progenies segregate in a ratio higher than 63:1. PCR analysis, performed with primers specific for NPTII and GUS genes, confirmed expression data. Moreover, unexpressed NPTII genes were identified in five and six progeny plants of two T1 genotypes. Recombinant phenotypes (GUS⁺ Km^S and GUS^- Km^R) were present in all progenies but the phenotype of our interest (GUS⁺ Km^S) were identified, up to now, in the progeny of only one T1 genotype. Southern blot analysis confirmed the transgenic nature of T1 genotypes and progeny plants. The performances and the efficiency of our transformation system will be compared with data reported in literature regarding other methods used to generate marker-free plants. The potential of the proposed system for the production of marker free plants in crop species will be discussed.