ISOLATION OF HOMOZYGOUS TRANSGENIC BRASSICA-NAPUS LINES CARRYING A SEED-SPECIFIC CHIMERIC 2S ALBUMIN GENE AND DETERMINATION OF LINKAGE RELATIONSHIPS

Using Agrobacterium tumefaciens-mediated gene transfer, 14 TO Brassica napus plants carrying one of three chimeric 2S albumin seed protein g enes were obtained after co-transformation with the neo gene. Plants w ere made homozygous using either cell haploid culture or, for single-c opy plants, traditional crossing methods. Sixty-five percent of kanamy cin-resistant plants contained at least one copy of the seed protein g ene, and multiple copies were usually at a single locus. In the majori ty of cases, at least one copy of the neo gene was linked to an introd uced 2S albumin gene, demonstrating that co-transformation is not a re liable way to obtain lines without the marker gene linked to the gene of interest. In 3 TO plants sequences derived from beyond the left bor der of the vector were integrated in the plant genome, in two cases pa rtially rearranged, confirming that T-DNA insertion is not always prec ise. Procedures for efficiently determining this are described. This w ork highlights the extra steps needed to prepare transgenic lines for field studies and potential further breeding.